JP7021230B2 - Compositions and Methods for Treating Inflammatory Diseases and Probiotic Compositions - Google Patents

Description

INDUSTRIAL APPLICABILITY The present invention has 3 for use in a method for treating an inflammatory disease, disorder or condition in a subject, particularly an inflammatory disease, disorder or condition of the gastrointestinal tract such as inflammatory bowel disease (IBD) and / or colorectal cancer. -Regarding hydroxybutyric acid (3-HB) or a salt thereof. The invention also relates to probiotic compositions comprising the compositions described herein and the use of such compositions for the treatment of gastrointestinal disorders.

Background Inflammation is a complex reaction of the immune system that involves the accumulation and activation of leukocyte and plasma proteins at the site of infection, toxic exposure or cytotoxicity. Inflammation also acts as a protective function in controlling infection and promoting tissue repair, but can also cause tissue damage and disease. IBD, such as Crohn's disease and ulcerative colitis, is associated with an abnormal intestinal inflammatory response. Uncontrolled inflammation also drives tumorigenesis in the intestinal tract, increasing the risk of developing colorectal cancer in patients with IBD.

It has been suggested that specific networks of cytokines and proteins are involved in the control of inflammation, especially inflammation of mucosal tissues such as the intestinal mucosa associated with the etiology of diseases such as IBD. To date drug therapies, including aminosalicylate and steroids, provide symptom improvement, but cannot stop the underlying inflammatory process and do not change the course of the disease.

Biologics, such as antibodies, offer alternative treatment options for these chronic diseases by targeting inflammatory cytokines or proteins.

The pro-inflammatory cytokine TNF-α is an example of a cytokine that plays an important role in inflammation, especially in intestinal inflammation (Neurath, M. F. (2014) Nature Reviews Immunology; 14: 329-342). TNF-α activates fibroblasts, stimulates pro-inflammatory cytokine production and angiogenesis, induces epithelial cell death, mediates T cell resistance to apoptosis, and induces cahexy. Anti-TNF-α agents have been the subject of numerous studies, many of which have been clinically approved for the treatment of inflammatory diseases such as psoriasis, Crohn's disease and rheumatoid arthritis (Neurath, MF (2014)). Nature Reviews Immunology; 14: 329-342). Examples include the monoclonal antibodies infliximab (Remicade®), adalimumab (Humira®), ertolizumab pegol (Simdia®) and golimumab (Simpony®).

IL-12 and IL-23 also have a role in inflammation and are particularly associated with intestinal inflammation (Teng, MWL, et al. (2015) Nature Medicine; 21: 719-729). IL-12 and IL-23 are induced in the inflamed mucosa of patients with Crohn's disease. IL-12 induces type 1 T helper ( _TH 1) cells, and Crohn's disease has been shown to be associated with _TH 1 response. Type 17 T helper ( _TH 17) cell responses have been identified in Crohn's disease and ulcerative colitis, and IL-23 is a well-known activator of _TH 17 cells. IL-12 and IL-23 share a subunit (p40), and the over-the-counter drug ustekinumab targets this subunit and is approved for Crohn's disease. Risankizumab is specific for the IL-23-specific p19 subunit, and Phase 2 data suggest efficacy in Crohn's disease (Neurath, MF (2017) Nature Reviews Gastroenterology &Hepatology; 14: 269-278). Other anti-IL-23 agents are in clinical development for the treatment of inflammatory diseases such as psoriasis, psoriatic arthritis, ankylosing spondylitis and rheumatoid arthritis.

The pro-inflammatory cytokine IL-6 also has a role in inflammation and is particularly associated with intestinal inflammation (Neurath, M. F. (2014) Nature Reviews Immunology; 14: 329-342). IL-6 activates T cells, blocks apoptosis, induces macrophage activation, recruits immune cells, activates acute phase proteins, induces epithelial cell proliferation, and supports tumor growth (Neurath). , MF (2014) Nature Reviews Immunology; 14: 329-342). Anti-IL-6 agents under development (eg tocilizumab) have shown early clinical efficacy in the treatment of Crohn's disease. In IBD patients, IL-6 and its agonist-soluble receptor sIL-6R are induced and intervene in T cell activation and its resistance to apoptosis. The IL-6 block is effective in experimental colitis (Neurath, M. F. (2017) Nature Reviews Gastroenterology &Hepatology; 14: 269-278). Anti-IL-6 agents are also under clinical development for the treatment of inflammatory diseases such as rheumatoid arthritis and are approved for the treatment of cancer.

Blocking IL-1β activity reduces tumorigenesis in mice by attenuating macrophage-dependent IL-6 secretion. Deficiencies in the IL-1β converting enzyme (ICE; also known as caspase 1) -an enzyme that cleaves IL-1β and IL-18 into active cytokines-protect mice from dextran sulfate sodium (DSS) -induced colitis. This suggests that blocking IL-1 family members may be appropriate for the treatment of chronic intestinal inflammation (Neurath, MF (2014) Nature Reviews Immunology; 14: 329-342).

IL-10 suppresses the production of pro-inflammatory cytokines by antigen-presenting cells and T cells and induces STAT3 signaling in regulatory T cells. IL-10 has been associated with inflammatory diseases, especially intestinal inflammation such as colitis. For example, IL-10 deficiency is associated with IBD (Neurath, M. F. (2014) Nature Reviews Immunology; 14: 329-342).

TGF-β1 is produced in many immune and non-immune cells of the mouse and human intestines, and two TGF-β1 receptors (types I and II) are expressed in virtually all intestinal cells. TGF-β1 suppresses the activation and function of effector T cells and macrophages, and regulates Foxp3-expressing T cells (Fantini, MC, et al. (2004) J Immunol; 172: _5149-5153 ) and TH 17 cells. Contributes to both peripheral differentiation. It also provides a chemotactic gradient for leukocytes and other cells that participate in the inflammatory response and, once the cells are activated, suppress it. TGF-β1 inhibits the production of extracellular matrix-degrading proteases by stromal cells and at the same time stimulates collagen production in these cells, promoting the margination of epithelial cells. TGF-β1 is a major cytokine involved in mucosal immunoglobulin A (IgA) production. Consistent with the data produced in a mouse model of inflammation, blocking endogenous TGF-β1 activity in the culture of normal, intestinal mucosal cells or explants and neutralizing antibodies enhances the induction of inflammatory molecules, while recombination. Stimulation of normal intestinal immune cells with TGF-β1 suppresses inflammatory signals. Monjasen is a drug under development for the treatment of Crohn's disease. Mongersen restores TGF-β1 activity by knockdown of the TGF-β1 inhibitor (Smad7) in mice, thereby suppressing the inflammatory pathway and eliminating colitis (Ardizzone, S., et al. (2016). ) Ther Adv Gastroenterol; 9 (4): 527-532).

Cytokines activate tumor cell proliferation, proliferation and survival through activation of intracellular signaling molecules such as STAT3 and NF-κB (Neurath, MF (2014) Nature Reviews Immunology; 14: 329-342). .. NF-κB is a protein complex that regulates DNA transcription, cytokine production and cell survival. Chronic mucosal inflammation in IBD is due to overactivation of effector immune cells that produce anti-levels of pro-inflammatory cytokines such as TNF-α, IL-6 and interferon-γ that result in colonic tissue damage. The nuclear transcription factor NF-κB has been identified as one of the key regulators in this immunological context. Trinitrobenzene sulfonic acid (TNBS) -induced colitis can be successfully treated by topical administration of p65 (subunit of NF-κB) antisense oligonucleotides, and the NF-κB pathway is an attractive target for therapeutic intervention in IBD. be. Like all cytokines and proteins involved in IBD, NF-κB is involved in the physiology of normal cells. NF-κB activation block in mouse hepatocytes has been associated with spontaneous onset of hepatocellular carcinoma, thus limiting NF-κB inhibition locally to immune cells within the inflamed colonic mucosa. Is desired (Atreya, I. et al. (2008) Journal of Internal Medicine; 263: 591-596).

Tissue remodeling and destruction in IBD is controlled by matrix metalloproteinase (MMP). Expression of MMP9 was found to be increased in patients with IBD, especially ulcerative colitis. In vivo animal studies indicate an important role for MMP9 in epithelial permeability disorders and inflammation enhancement (Neurath, M. F. (2017) Nature Reviews Gastroenterology &Hepatology; 14: 269-278).

Biopharmacy that targets cytokines and proteins, such as monoclonal antibodies, are highly target-specific by their very nature. Antibody therapy is often associated with secondary failure due to long-term intolerance and drug withdrawal (Amiot, A. et al. (2015) Ther Adv Gastroenterol; 8 (2): 66-82).

Miyarisan Pharmaceutical Co., Ltd. (Japan) produces Clostridium butyricum (CBM 588 strain) probiotics for gastrointestinal health. This product uses a non-GMO Clostridium strain that does not produce 3-HB. Clostridium butyricum was found in the human gut microbiota and has a proven track record of safe use as a probiotic for human and animal health.

There is still a need for novel therapeutic agents in the art to treat inflammatory diseases, disorders or conditions, particularly inflammatory diseases, disorders or conditions in the intestinal tract, including colorectal cancer. There is still a need in the art for therapeutic agents capable of suppressing the inflammatory process underlying the disease, disorder or condition and / or altering the course of the disease, disorder or condition. More effective; less side effects; orally acceptable; easier to administer, improved compliance with treatment schedules; and / or targeted for treatment of local diseases, disorders or conditions at the site of inflammation There is still a need for modified therapeutic agents in the field.

Abstract of the Invention In one aspect, the invention provides 3-hydroxybutyric acid (3-HB) or a salt thereof for use in a method of treating an inflammatory disease, disorder or condition in a subject.

In a second aspect, the invention provides a pharmaceutical composition comprising 3-HB or a salt thereof for use in a method of treating an inflammatory disease, disorder or condition in a subject.

In a third aspect, the invention is a pharmaceutical composition for use in a method of treating an inflammatory disease, disorder or condition in a subject, wherein the composition comprises a 3-HB delivery means, the method. Includes delivering 3-HB delivery means to the lumen and / or mucosal surface of the gastrointestinal (GI) tract.

In a fourth aspect, the invention comprises subjecting a) 3-hydroxybutyric acid (3-HB) or a salt thereof; b) a pharmaceutical composition comprising 3-HB or a salt thereof; and / or c) 3-HB delivery. Provided are methods of treating an inflammatory disease, disorder or condition in a subject, comprising administering a pharmaceutical composition comprising means.

In a fifth aspect, the invention provides a composition comprising or essentially consisting of a genetically engineered anaerobic bacterium producing (R) -3-HB and an orally ingestible carrier.

In a sixth aspect, the invention provides a method of treating or preventing a gastrointestinal disease or disorder comprising administering a genetically engineered anaerobic bacterium that produces (R) -3-hydroxybutyrate. ..

In a seventh aspect, the invention provides a method of treating or preventing gastrointestinal flora abnormalities, comprising administering a genetically engineered anaerobic bacterium that produces (R) -3-hydroxybutyrate. ..

In an eighth aspect, the invention provides a method of treating or preventing Clostridium difficile infection, comprising administering a genetically engineered anaerobic bacterium that produces (R) -3-hydroxybutyrate.

In a ninth aspect, the invention provides a method of regulating the intestinal flora in a subject, comprising administering a genetically engineered anaerobic bacterium that produces (R) -3-hydroxybutyrate.

FIG. 1A shows the natural acid production metabolic pathway in Clostridium.

FIG. 1B shows the acid-producing metabolic pathway in Clostridium after the introduction of unnatural (R) -3-hydroxybutyryl-CoA dehydrogenase.

FIG. 2 shows a codon-optimized DNA sequence for the phaB gene from Cupriavidus necator.

FIG. 3 details a plasmid map of pfdx_phaB in pMTL83251 (Clostridium butyricum).

FIG. 4A shows the temporal production of (R) -3-HB, butyrate and acetate by wild-type Clostridium butyricum (wt).

FIG. 4B shows the temporal production of (R) -3-HB, butyrate and acetate by genetically engineered Clostridium butyricum (CHN-1-phaB).

FIG. 5 shows total CFU (A) and spores (heat resistance CFU) produced by wild-type Clostridium butyricum and genetically engineered Clostridium butyricum (CHN-1-phaB) measured as CFU / mL over time. B) is shown.

FIG. 6 shows the percentage of spores to vegetative cells produced by wild-type Clostridium butyricum and genetically engineered Clostridium butyricum (CHN-1-phaB) over time.

FIG. 7 shows the total number of viable cells of CHN-1 on modified BIM expressed as colony forming units / mL. CFU / mL is shown as the average of three independent experiments and error bars represent the standard deviation.

FIG. 8 shows the number of heat resistant cells on modified BIM expressed as colony forming units / mL. CFU / mL is shown as the average of three independent experiments and error bars represent the standard deviation.

FIG. 9 shows the pH in the simulated colon measured at various time points.

FIG. 10 shows the presence of acetate (mM) in the simulated colon at the time of selection.

FIG. 11 shows the presence of butyrate (mM) in the simulated colon at the time of selection.

FIG. 12 shows the production of (R) -3-HB in the simulated colon at 24 hours for the compound experiment repeat (A) and each experiment repeat (B).

FIG. 13 shows a 16s-23s intergenic spacer region-specific PCR using oligonucleotides ISR-F and ISR-R to detect CHN-1 in a bioreactor.

FIG. 14 shows a phaB-specific PCR using oligonucleotides phAB-F and phaB-R for detecting CHN-1 in a bioreactor.

FIG. 15 shows data from an intestinal organoid model. The relative expression (mRNA) levels of the inflammatory factors NF-κB (A) and TNFα (B) to unstimulated organoids when the organoids were incubated with TNFα, TNFα butyrate and / or (R) -3-HB. offer.

FIG. 16 shows data from an intestinal organoid model. Inflammatory markers IL-10 (A), IL-23 (B), TNF-α (C), IL-1β (when the organoids are stimulated with TNF-α alone or in combination with (R) -3-HB) D), TGF-β1 (E), IL-6 (F) and NF-κβ (G) are shown relative expression (mRNA) to unstimulated organoids.

FIG. 17 shows the relative mRNA expression levels of IL-23 in organoids treated with 60 ng / mL TNF-α and increasing concentrations of butyrate or (R) -3-HB.

FIG. 18 shows data from an exobibo tissue model. Inflammatory markers IL-10 (A), IL-12 (B), IL-1β (C) and IL when exobibo colon tissue samples are stimulated with TNF-α alone or in combination with (R) -3-HB. The amount of protein (Luminex) relative to the unstimulated tissue sample for -6 (D) is shown.

FIG. 19 shows data from an exobibo tissue model. Comparative Protein Dot Blot Array experiments were used to compare protein expression levels in exobibo colon tissue samples stimulated with TNF-α alone or with (R) -3-HB. The data of the inflammation markers TNF-α (A), IL-23 (B) and MMP9 (C) are shown.

FIG. 20 shows CFU / mL obtained by germination and elongation of Clostridium butyricum and Clostridium difficile spores, either alone or in co-culture.

FIG. 21 shows colony forming units per mL of CHN-1 spores on an RCM agar plate after incubation under gastric and small intestinal conditions. Data points represent the average of three independent experiments, and error bars represent the standard deviation.

Detailed Description The present invention described herein is a large variety of 3-HBs, in particular (R) -3-hydroxybutyrate ((R) -3-HB) produced from intestinal cells. Based on the surprising findings of the inventors of the present application that they are potent anti-inflammatory agents that act on inflammatory cytokines and signaling molecules. In particular, we found that 3-HB is a specific pro-inflammatory cytokine and protein (eg, TNF-α, IL-23, IL-6, IL-1β, IL-12 and) in inflamed intestinal tissue. It was shown to down-regulate MMP9) and up-regulate certain anti-inflammatory cytokines (eg IL-10, TGF-β1). The involved cytokines and proteins have been associated with a number of inflammatory diseases, disorders or conditions, especially inflammation in the gastrointestinal tract, including IBD (eg Crohn's disease, ulcerative colitis and colorectal cancer).

The present invention meets the above needs in the art. In particular, the present invention provides improved treatments for treating inflammatory diseases, disorders or conditions that have broad effects on a large number of different inflammatory cytokines and proteins.

We have shown that 3-HB exhibits an anti-inflammatory effect when delivered topically to inflamed tissue, especially intestinal tissue. The present invention therefore provides an improved therapeutic agent for the treatment of an inflammatory disease, disorder or condition that can be delivered topically to the site of inflammation.

The present invention provides improved therapeutic agents for the treatment of inflammatory diseases, disorders or conditions that suppress and / or alter the underlying inflammatory process.

In certain cases, the invention provides prophylactic treatment or treatment of colorectal cancer.

In particular, the invention provides improved treatment for inflammatory diseases, disorders or conditions compared to biologics such as antibodies that target individual cytokines. In particular, 3-HB has broader effects on a large number of different inflammatory cytokines and proteins in inflamed tissues as compared to such biologics.

In certain embodiments, the present invention provides a pharmaceutical composition that delivers 3-HB to the gastrointestinal tract.

The intestinal flora is associated with the promotion of colon health. One mechanism believed to be achieved by this is through the production of short chain fatty acid (SCFAs) acetate, propionate and butyrate by dietary fiber fermentation. Butyrate has received the most attention as an important mediator of its effects on colon health, especially its anti-inflammatory and antitumor effects. Intestinal microbiome analysis confirms a significant reduction in butyrate-producing bacteria in the colon of patients with ulcerative colitis and colon cancer (Frank, DN, et al. (2007) Proc Natl Acad Sci; 104 ( 34): 13780-13785; Wang, T., et al. (2012) The ISME Journal; 6: 320-329). Colon irrigation with butyrate has been shown to reduce inflammation in ulcerative colitis (Hamer, H. M., et al. (2008) Aliment Pharmacol Ther; 27: 104-119).

We show that 3-HB reduces the relative expression levels of some pro-inflammatory cytokines and proteins to an extent greater than butyrate in inflamed tissues, especially intestinal tissues, and anti-inflammatory cytokines. It has been shown to increase the relative expression level of. In particular, (R) -3HB shows a larger effect of reducing IL-23 expression than butyrate at a low concentration (10 to 100 μM).

(R) -3-HB, also known as D-β-hydroxybutyrate (β-OHB), is a site that is naturally produced in the liver and can enter the bloodstream and act as a metabolic substrate during carbohydrate restriction. It is a ketone body that is circulated in the extrahepatic tissue. (R) -3-HB also functions in various signaling pathways, but is not normally found in the adult intestinal lumen. Ingestion of 3-HB in dissociated (acid) form is impractical. The acid is formulated as a salt or ester for ingestion, but in the scenario described above, 3-HB is rapidly absorbed into the small intestine, enters the bloodstream, where it is diluted and distributed systemically. Oral administration of a salt of 3-HB is unsuitable because the formulation results in potentially dangerous high salt concentrations (eg, sodium salts).

The present invention provides a pharmaceutical composition that can be administered enterally, preferably orally. The present invention therefore provides an orally acceptable and well tolerated pharmaceutical composition for 3-HB. The pharmaceutical composition exhibits improved patient compliance, for example, as compared to compliance with a ketone-induced diet.

3-HB can be delivered to the intestine, preferably to the anaerobic portion of the intestine, preferably to the large intestine, preferably to the colon. 3-HB can be delivered so that it is not absorbed in the small intestine. Preferably, 3-HB is not delivered to the esophagus, stomach or small intestine. The present invention also provides a pharmaceutical composition that can be administered enteral, preferably rectum.

3-HB can therefore be delivered to the site of inflammation, especially the lumen of the gastrointestinal tract, where it exerts its action locally. Therefore, adverse side effects are minimized or avoided. In particular, adverse side effects associated with delivery and systemic uptake of therapeutically effective amounts of ketone bodies (eg, salt overload) are minimized or avoided.

3-HB is a chiral compound with the diisomers (R) -3-HB and (S) -3-HB. The 3-HB of the present invention can be an individual isomer, a racemic mixture of isomers or a non-racemic mixture of isomers. The racemic mixture of (R) -3-HB and (S) -3-HB has about 50% by weight (R) -3-HB and about 50% by weight (S) -3-HB. Alternatively, at least about 50% by weight, 60% by weight, 70% by weight, 80% by weight or 90% by weight of 3-HB can be (R) -3-HB and the rest can be (S) -3-HB. .. Preferably, substantially all or 100% by weight of 3-HB can be (R) -3-HB.

The molar ratio of (R) -3-HB to (S) -3-HB can be greater than 5: 1, greater than 10: 1, greater than 50: 1, or greater than 100: 1. In certain embodiments, the ratio of (R) -3-HB to (S) -3-HB is about 100-5: 1, 100-50: 1, 100-20: 1, 50-5: 1, 20. It can be in the range of ~ 5: 1, 15-5: 1 or about 15-10: 1.

3-HB is commercially available as a pure enantiomer in the form of (R) or (S) or a racemic mixture of (R) -3-HB and (S) -3-HB. 3-HB can also be produced by methods known in the art. Preferably, 3-HB can be produced by fermentation of anaerobic bacteria genetically engineered to produce 3-HB. 3-HB can be isolated by methods known in the art. Preferably, 3-HB can be produced by fermentation of the novel Clostridium strains described herein that produce chiral compounds. For example, a Clostridium species, preferably Clostridium, comprising a heterologous gene capable of expressing (R) -3-hydroxybutyryl-CoA dehydrogenase from 3-HB, which can be 100% by weight (R) -3-HB. It can be produced by fermentation of butyricum). Increased titers can be achieved by co-introduction of heterologous genes capable of expressing butyrate kinase and phosphotransbutylylase. Introduction of a heterologous gene capable of expressing (R) -3-hydroxybutyryl-CoA dehydrogenase results in the production of the (R) form of 3-hydroxybutyryl-CoA. The natural reductase enzyme then converts (R) -3-hydroxybutyryl-CoA to (R) -3-HB. Alternatively, a heterologous gene capable of expressing (R) -3-hydroxybutyryl-CoA dehydrogenase with 3-HB which can be at least about 90% by weight (R) -3-HB and the rest being (S) -3-HB. It can be produced by fermentation of Clostridium species, preferably Clostridium butyricum. An increase in 3-HB titer can be achieved by introducing a heterologous gene capable of expressing propionyl-CoA transferase (PCT). Introduction of the heterologous (R) -3-hydroxybutyryl-CoA dehydrogenase and propionyl-CoA transferase genes results in the production of (R) -3-HB and (S) -3-HB in an approximately 10: 1 ratio.

3-HB can be in the form of a pharmaceutically acceptable salt or solvate. As used herein, "3-HB" refers to 3-HB or a salt thereof. As used herein, "pharmaceutically acceptable salt" refers to any salt preparation suitable for use in pharmaceutical applications. Pharmaceutically acceptable salts are amine salts such as N, N'-dibenzylethylenediamine, chloroprocine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, prokine, N-benzyl. Phenethylamine, 1-para-chloro-benzyl-2-pyrrolidin-1'-ylmethylbenzoimidazole, diethylamine and other alkylamines, piperazine, tris (hydroxymethyl) aminomethane, etc .; alkali metal salts such as lithium, potassium, sodium Etc; alkaline earth metal salts such as barium, calcium, magnesium; transition metal salts such as zinc, aluminum; other metal salts such as sodium hydrogen phosphate, disodium phosphate etc .; mineral acids such as hydrochloride, etc. Such as sulfates; and includes salts of organic acids such as acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, fumarate and the like.

Although it is possible to administer 3-HB by itself, it is preferable to include 3-HB in the pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition comprising 3-HB or a salt thereof for use in a method of treating an inflammatory disease, disorder or condition in a subject. Preferably, the pharmaceutical composition is formulated to deliver 3-HB to the gastrointestinal tract by releasing 3-HB in the lumen or mucosal surface of the gastrointestinal tract.

Alternatively, the pharmaceutical composition can deliver 3-HB to the lumen or mucosal surface of the gastrointestinal tract by other means. Accordingly, the present invention is a pharmaceutical composition for use in a method of treating an inflammatory disease, disorder or condition in a subject, wherein the composition comprises a 3-HB delivery means and the method is 3-. It comprises delivering the HB delivery means to the lumen or mucosal surface of the gastrointestinal tract.

"3-HB delivery means" can mean any chemical or biological means for delivering 3-HB or a salt thereof to the lumen of the gastrointestinal tract. Suitable examples include a biological delivery system that delivers a 3-HB prodrug or 3-HB to the lumen of the gastrointestinal tract. Such means of delivery are further described below.

The present invention comprises a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and optionally other therapeutic and / or prophylactic ingredients.

The pharmaceutical compositions of the invention are administered so that a therapeutically effective amount of 3-HB is delivered and in any acceptable dosage form of a drug of similar use.

Pharmaceutical compositions include those suitable for oral or rectal administration. Preferably, the administration is oral using a convenient daily dose regimen that can be adjusted according to the degree of distress.

The pharmaceutical composition of the present invention may be prepared with one or more conventional adjuvants, carriers or diluents and made into dosage forms such as unit doses. The pharmaceutical composition and dosage form may contain a conventional ingredient in a conventional ratio, wherein the dosage form is an effective amount of active agent (3-HB described herein) appropriate for the intended daily dose range to be used. ) Can be included.

The pharmaceutical composition may take any of various forms, in particular, depending on the embodiment in which it is used. Therefore, the composition may be administered to humans or animals in need of the form or treatment of powders, tablets, capsules, solutions, creams, gels, hydrogels, foams, micelle solutions, liposome suspensions. It can be any other suitable form. The carrier of the pharmaceutical composition of the present invention must be sufficiently tolerant of the subject to be administered.

The "pharmaceutically acceptable carrier" used herein is any known compound or combination of known compounds known to those of skill in the art to be useful in the formulation of pharmaceutical compositions.

Activators can be used in monotherapy (ie, use of activators alone) for the treatment of inflammatory diseases, disorders or conditions in a subject. Alternatively, the activator may be 5-aminosalicylate (5-ASAs), such as 5-ASA, mesalazine, sulfasalazine, orsalazine and balsalazine, especially Asacol HD®, Delzicol®, Pentasa®. , Rialda® and Apriso®; steroids, especially corticosteroids such as hydrocortisone, methylpredonizolone, prednison, prednisolone and budesonide; immunomodulators including immunosuppressants such as azathiopurine, 6-mercaptopurine, metuximab, Cyclosporin-A and tachlorimus; biological agents including anti-TNF agents such as infliximab, adalimumab, golimumab and sertrizumab pegor, T cell transfer agents such as integrin blocker natalizumab and vedrizumab, antibiotics and non-pathogenic microorganisms such as Probiotics including symbiotic Escherichia coli, lactobacillus, saccharomyces or parasite pig whip; chemotherapeutic agents for colorectal cancer such as capecitabin (Xeloda®), fluorouracil (5-FU, Adolcil®) ), Irinotecan (Camptosal®), Oxaliplatin (Eloxatin®) and Trifluridine / Tipiracil (TAS-102, Ronsurf®); Targets for colorectal cancer including anti-angiogenic therapeutic agents Chemical therapeutic agents such as bevasizumab (Avastin®), legoraphenib (Stiberga®), ziv-aflibercept (Zaltrap®) and ramsilmab (Silamza®) and epithelial cell proliferation factor receptors. (EGFR) inhibitors such as cetuximab (Arbitux®) and panitumumab (Vectibix®); as an adjunct to one or more additional active agents including short chain fatty acids such as butyrate, such as butyric acid, or with it. Can be used in combination. 3-HB can be used in combination with any one or more of the above and, for example, azathioprine and infliximab. Preferably, 3-HB can be used in combination with butyrate.

In certain preferred embodiments, the pharmaceutically acceptable carrier may be a solid and the composition may be in the form of a powder or tablet. Solid pharmaceutically acceptable carriers include flavoring agents, buffers, lubricants, stabilizers, solubilizers, suspending agents, wetting agents, emulsifying agents, dyes, fillers, flow promoters, compression aids. , May contain one or more substances that can also act as inert binders, sweeteners, preservatives, pigments, coatings or tablet disintegrants. The carrier can also be a capsule filler. In the powder, the carrier is a fine powder solid mixed with the fine powder activator of the present invention. In tablets, the activator can be mixed with a carrier having the required compressive properties in an appropriate ratio and compressed to the desired shape and size. Powders and tablets may preferably contain up to 99% activator. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidin, low melting point wax and ion exchange resins. In other embodiments, the pharmaceutically acceptable carrier may be a gel and the composition may be in the form of a cream or the like.

The carrier may contain one or more additives or diluents. Examples of such additives are gelatin, gum arabic, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide and the like.

However, in other preferred embodiments, the pharmaceutically acceptable carrier may be a liquid and the pharmaceutical composition may be in the form of a solution. Liquid carriers are used in the production of solutions, suspensions, emulsions, syrups, elixirs and pressure compositions. The activator of the invention may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture thereof or a pharmaceutically acceptable oil or fat. Liquid carriers are other suitable such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavors, suspensions, thickeners, colorants, viscosity modifiers, stabilizers or osmoregulators. May contain various pharmaceutical additives. Suitable examples of liquid carriers for oral administration are water (partially containing the above additives such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monovalent and polyhydric alcohols such as glycols). And their derivatives and oils (eg fractionated coconut oil and peanut oil).

The pharmaceutical compositions of the present invention include other solutes or suspensions (eg, saline or glucose sufficient to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (eg, polysorbate 80). It can be orally administered in the form of a sterile solution or suspension containing sorbitol copolymerized with ethylene oxide and an oleic acid ester of its anhydride). The agents used according to the invention are also orally administered in liquid or solid composition form. Compositions suitable for oral administration include liquid forms such as syrups, elixirs and suspensions, such as capsules containing solid and liquid forms such as pills, capsules, granules, tablets and powders. All known to those in the art.

The pharmaceutical composition of the present invention may also be formulated for rectal administration, including suppositories and enema formulations. For suppositories, a low melting point wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active ingredient is uniformly dispersed, for example by stirring. The molten homogeneous mixture is then poured into a convenient sized mold, cooled and solidified. Enema formulations may be in semi-solid or suspension containing gel or ointment, liquid form containing aqueous solution or foam, which will be known to those of skill in the art.

Other suitable pharmaceutical carriers and their formulations are described in Remington: The Science and Practice of Pharmacy 22nd Edition, The pharmaceutical Press, London, Philadelphia, 2013.

The pharmaceutical composition of the present invention can be formulated as a release-regulating dosage form. "Release control" means that the dosage form is a formulation in which the release rate and / or site of the active agent is different from that of the immediate release dosage form administered by the same route. This modification is achieved by a special pharmaceutical design and / or manufacturing method. Release control forms include oral dose release control forms. A sustained release (or sustained release) dosage form is a release-regulating dosage form that exhibits sustained release over a long period of time. In the delayed release dosage form, the release of the active substance is delayed for a period of time after administration or application of the drug (delay is also known as lag time). Subsequent release may resemble an immediate release dosage form. Polyphase release dosage forms include two-phase release and pulsed release. In the two-phase release dosage form, the first phase of drug release is determined by the partial dose that provides the therapeutic agent level immediately after administration, and the second sustained release phase is the partial dose required to maintain long-term effective therapeutic levels. offer. Pulsed drug release is intended to deliver the drug instantaneously at specific time intervals. Multi-unit: The multi-unit dosage form comprises multiple units, eg pellets or beads, each containing a release control additive, contained in gelatin capsules or compressed into tablets. 1 unit: 1 unit The dosage form consists of only 1 unit, eg osmotic tablets.

Release control additives and formulations are well known in the art and certain technologies are commercially available.

Suitably, the pharmaceutical compositions of the invention are preferably formulated to deliver 3-HB to the gastrointestinal tract by oral administration. The human gastrointestinal tract extends from the mouth to the anus and consists of a digestive structure that includes the esophagus, stomach and intestines. The gastrointestinal tract does not include attached glandular organs such as the liver, bile ducts or pancreas. The intestine includes the small and large intestines. The small intestine includes the duodenum, jejunum and ileum. The large intestine includes the cecum, colon, rectum and anus. The upper gastrointestinal tract includes the buccal oral cavity, pharynx, esophagus, stomach and duodenum. The lower gastrointestinal tract contains the small and large intestines below the duodenum. Preferably, the pharmaceutical composition of the present invention delivers 3-HB to the lumen or mucosal surface of the gastrointestinal tract, more preferably to the lumen or mucosal surface of the large intestine, more preferably to the lumen or mucosal surface of the colon. .. Preferably, the pharmaceutical composition of the invention delivers 3-HB to the anaerobic portion of the gastrointestinal tract, preferably the colon and / or the terminal small intestine (ileum).

Covalent bonding of drugs and carriers, including those that increase stability and hydrophilicity; coating with pH-sensitive polymers; sustained release formulations; development of carriers that are particularly degraded by colonic bacteria; bioadhesive systems; And various strategies have been proposed for colon targeting of orally administered drugs, including osmotic control drug delivery systems. Various prodrugs have been developed for the delivery of 5-aminosalicylic acid (5-ASA) for topical treatment of IBD. Microbiologically degradable polymers, especially azo cross-linked polymers, are being studied for use as coatings for colon-targeted drugs. Certain plant polysaccharides such as amylose, inulin, pectin and guar gum remain unaffected by gastrointestinal enzymes and are being sought as drug coatings for the formulation of colon-targeted drug delivery systems. In addition, the combination of plant polysaccharides and crustacean extracts containing chitosan or its derivatives is of interest in the development of colonic delivery systems.

An example of an additive for a release-regulated formulation is a hydrogel, which can rapidly swell in water and retain large amounts of water in its swelling structure. Different hydrogels can provide different drug release patterns and the use of hydrogels to promote colonic delivery has been studied. For example, hydrogels and xerogels are manufactured using the highly viscous acrylate gel, Eudispert hv, which has excellent retention in the lower rectum over time. Evonik Industries, Evonik Industries, provides various forms of coating, including gastric resistance, pH controlled drug release, colon delivery, activity and protection from activity.

The pharmaceutical composition is a mixture of, for example, 3-HB, one or more pharmaceutically acceptable carriers, additives and / or diluents and one or more release control additives by any of the techniques known in the art. Can be manufactured by The pharmaceutical composition comprises 3-HB and one or more pharmaceutically acceptable carriers, additives and / or diluents and optionally one or more release control additives using techniques known in the art. It can be manufactured by coating the core with a release control layer or coating. For example, the coating can be formed by crimping using any of the known press coaters. Alternatively, the pharmaceutical composition can be produced by granulation and agglomeration techniques or can be constructed by spray drying techniques followed by drying.

The thickness of the coating can be tightly controlled using any of the above techniques. One of ordinary skill in the art can select the thickness of the coating as a means to obtain the desired rate at which the drug is released after the desired lag time and / or lag time.

In the pH-dependent system, the pH of the human gastrointestinal tract is from the stomach (pH can be about 1-2 and rises to pH 4 during digestion) to the small intestine where digestion is located (pH is about 6-7). Follow the general knowledge that it gradually rises through) and then rises in the distal ileum. Coated tablets, capsules or pellets with pH sensitive polymers provide delayed release and protect the active drug from gastric fluid.

The pharmaceutical composition of the present invention may be formulated to deliver 3-HB to the gastrointestinal tract at a specific pH. Commercially available additives include Eudragit® polymers that can be used to deliver 3-HB to specific locations in the gastrointestinal tract. For example, the pH in the duodenum can exceed about 5.5. Eudragit® L 100-55 (powder), Eudragit® L 30 D-55 (aqueous dispersion) and / or Acryl-EZE® (powder), eg Eudragit® L Based on 100-55, it can be used as a ready-to-use enteric coating. The pH of the jejunum is from about 6 to about 7, and Eudragit® L 100 (powder) and / or Eudragit® L 12,5 (organic solution) can be used. Delivery to the colon can be achieved at pH above about 7.0, Eudragit® S 100 (powder), Eudragit® S 12,5 (organic solution) and / or Eudragit® FS. 30 D (aqueous dispersion) can be used. Plas ACRYL ^TM T20 flow promoter and plasticizer premixes specifically designed for Eudragit® FS 30 D formulations may also be used.

The pharmaceutical composition, 3-HB, about 5.5 or more, for example about 5.6, 5.7, 5.8 or 5.9 or more; preferably 6 or more, for example about 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9 or higher; preferably 7 or higher, for example about 7.1, 7.2, 7.3, 7. It may be formulated to be delivered at a pH of 4, 7.5, 7.6, 7.8, 7.9 or 8. Preferably, the pharmaceutical composition may be formulated to deliver 3-HB at a pH of about 5.5-7, about 6-7.5 or 7-8. In certain embodiments, the pharmaceutical composition releases 3-HB or 3-HB delivery means at an appropriate pH, thus delivering 3-HB to the lumen of the gastrointestinal tract, preferably the terminal ileum and / or colon. do.

The pharmaceutical composition taken on an empty stomach may reach the ascending colon about 5 hours after administration, and the actual arrival depends largely on the rate of gastric emptying. Drug delivery into the colon is greatly affected by the rate at which it travels through this area. In healthy men, capsules and tablets pass through the colon in an average of 20-30 hours. Solutions and particles are usually widespread within the proximal colon and are often dispersed throughout the large intestine.

The pharmaceutical compositions of the present invention may be formulated for time-controlled delivery to the gastrointestinal tract, i.e., to deliver 3-HB after a period of time (lag time).

Commercially available additives for time-controlled delivery are Eudragit® RL PO (powder), Eudragit® RL 100 (granule), Eudragit® RL 30 D (aqueous dispersion). Body) and Eudragit® RL 12,5 (organic solution). These additives are insoluble, highly permeable, pH-independent swelling additives that can provide a customized release profile by combining Eudragit® RS in various proportions. Eudragit® RS PO (powder), Eudragit® RS 100 (granule), Eudragit® RS 30 D (aqueous dispersion) and Eudragit® RS 12,5 (organic solution) , Eudragit® RL combined in various proportions to provide a customized release profile, an insoluble, low permeability, pH independent swelling additive. Eudragit® NE 30 D (Aqueous Dispersion), Eudragit® NE 40 D (Aqueous Dispersion) and Eudragit® NM 30 D (Aqueous Dispersion) can be matrix-forming agents. It is an insoluble, low-permeability, pH-independent swelling additive.

Preferably, the pharmaceutical composition may be formulated to deliver 3-HB to the gastrointestinal tract approximately 4 hours after administration. Preferably, the pharmaceutical composition comprises 3-HB about 4 to 48 hours after administration, preferably about 5 to 24 hours after administration, for example, about 5 hours, 10 hours, 15 hours, 20 hours or 24 hours after administration. It may be formulated to be delivered preferably after about 5-10 hours, 5-15 hours, 5-20 hours or about 10-24 hours, 15-24 hours or 20-24 hours after administration. Preferably, the pharmaceutical composition is administered between or after a meal, preferably after a meal. In certain embodiments, the pharmaceutical composition releases 3-HB after a lag time. Alternatively, the pharmaceutical composition releases the 3-HB delivery means after a lag time.

Release of 3-HB or 3-HB delivery means from the pharmaceutical composition at appropriate pH or after lag time can be either immediate release or release regulation. Immediate release and release control formulations are known to those of skill in the art.

Release of 3-HB or 3-HB delivery means from a pharmaceutical composition can be measured by methods known in the pharmaceutical industry. Drug dissolution tests provide critical in vitro drug release information for both quality control purposes (to assess batch-to-batch consistency of solid oral dosage forms such as tablets) and drug development (to predict in vivo drug release profiles). It is used on a daily basis to do so. The dissolution test was performed at USP Dissolution FIGURE 1-basket (37 ° C); USP Dissolution FIGURE 2-paddle (37 ° C); USP Dissolution FIGURE 3-reciprocating cylinder (37 ° C); USP Dissolution FIGURE 4-flow-through cell (37 ° C). ) Can be performed in a melting device.

Preferably, 3-HB is substantially not released from the pharmaceutical composition until the appropriate pH is reached and / or the lag time elapses. Preferably, the 3-HB delivery means is substantially not released from the pharmaceutical composition until the appropriate pH is reached and / or the lag time elapses. Preferably, the 3-HB or 3-HB delivery means released from the pharmaceutical composition is less than 10% by weight, preferably from the pharmaceutical composition, until the appropriate pH is reached and / or the lag time elapses. The released 3-HB or 3-HB delivery means is 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight or less than 1% by weight. be.

In certain embodiments, the pharmaceutical composition can be formulated, preferably as a tablet, using Multi Matrix MMX® technology (Cosmo Pharmaceuticals Inc.). Tablets made with MMX® technology are coated with a pH resistant acrylic copolymer that delays release until the tablet reaches the indicated intestinal region where the programmed lysis begins. The active agent is protected from adverse pH conditions and enzymes present in the upper gastrointestinal tract. Regulation of release over the length of the colon not only simplifies application to the patient, but also allows topical application of the active pharmaceutical ingredient to the surface affected by inflammation. For example, the pharmaceutical composition can be formulated as Zacol NMX® (Cosmo Pharmaceuticals Inc.) and the tablets are calcium 3-HB, maltodextrin, inulin, sorbitol, hypromellose, microcrystalline cellulose, processed corn starch, citric acid, colloid. Silica hydrate, talc, shelac, magnesium stearate, stearate, lecithin, titanium dioxide, hydroxypropyl, triethylcitrate; aroma: vanillin is included.

In another embodiment, the pharmaceutical composition comprises a capsule shell containing 3-HB, hydroxypropylmethyl cellulose buffered with calcium and magnesium (3-hydroxybutyric acid, calcium hydroxide, magnesium hydroxide and medium chain triglyceride). It can be formulated as a BioCare® capsule containing the anti-solidification agent silicon dioxide and magnesium stearate. The capsule is about 2.3 cm in length.

The pharmaceutical composition is an invasive medium for aesthetic purposes (eg, colorants), for stability purposes (eg, coated with a moisture-proof layer), for flavoring purposes or for 3-HB, prodrugs, delivery systems and / or additives. It can be overcoated with a pharmaceutically acceptable film coating for protection from. Preferably, the pharmaceutical composition is gastroprotective or enteric, represented, for example, a mixture of acrylic acid and / or methacrylic acid copolymer types A and / or type B (eg, as Eudragit S100 and / or Eudragit L100). Can be overcoated with coating. Preferably, the mixture of acrylic acid and / or methacrylic acid copolymer type A and / or type B is in the range of 1: 5-5: 1. The gastroprotective coating also optionally comprises a plasticizer, a dye, at least one water-solvent, at least one organic solvent or a mixture thereof.

"Prodrug" means a derivative of a drug molecule that needs to be converted in the body to release the active drug. Colon drug delivery strategies include the use of prodrugs, which are metabolized by enzymes found only in the colon.

Biological Delivery System In certain embodiments, a pharmaceutical composition that delivers 3-HB to the gastrointestinal tract comprises a biological delivery system capable of producing 3-HB.

"Biological delivery system" means a biological agent, preferably a bacterial agent, such as a microbial agent that can be orally administered and can produce 3-HB. Preferably, the biological delivery system can be a genetically engineered anaerobic bacterium capable of producing 3-HB. The bacterium can produce 3-HB as the only fermentation product or in combination with short chain fatty acids (SCFAs) such as acetate and / or butyrate.

The compositions of the invention may include genetically engineered anaerobic bacteria and orally ingestible carriers that produce 3-HB. The composition delivers 3-HB to the subject. When taken orally, the bacterium substantially grows within the subject, producing 3-HB and secreting it into the anaerobic portion of the gastrointestinal tract. The bacterium secretes 3-HB as it passes through the intestine or when it begins to attach to the epithelial / mucosal cell wall underlayer.

The bacterium can be an anaerobic bacterium. Anaerobic bacteria are bacteria that can survive in an oxygen-limited (hypoxic) or completely oxygen-depleted (anoxic) environment. These are obligate anaerobes, bacteria that are harmed by the presence of oxygen and can only grow in anaerobic (anoxic) environments; they can survive in aerobic environments (aerobic), but are the final electron acceptors in the respiratory pathway. Air-tolerant bacteria that cannot use molecular oxygen as; and can survive in both aerobic and anaerobic environments, and due to the availability of favorable electron acceptors, use molecular oxygen or other molecules as the final electron acceptor in the respiratory pathway. Contains facultative anaerobic bacteria that can. Preferably, the bacterium is an obligate anaerobic bacterium.

In certain embodiments, the bacterium is Clostridia. Introduction of an unnatural gene capable of expressing (R) -3-HB dehydrogenase ((R) -3-HBD) results in a Clostridium strain capable of producing (R) -3-HB. The engineered Clostridia produces (R) -3-hydroxybutyryl-CoA. Natural PTB and BUK enzymes, if present, can convert (R) -3-hydroxybutyryl-CoA to (R) -3-HB. (R) -3-HB is secreted into the intestine.

Clostridia, which naturally produces butyrate as the main fermentation product, are now adapted to produce (R) -3-HB in place of or in combination with butyrate. Clostridia can also produce other useful fermentation products such as acetates, propionates, vitamins and bacteriocins.

Bacteria that are part of the natural intestinal flora, i.e., those found naturally in the intestine, are preferred. Bacteria that naturally produce butyrate are also preferred.

Clostridia is the preferred bacterial class for inclusion in the composition. The Clostridia family includes Clostridiaceae, Christensenellaceae, Eubacteriaceae, Lachnospiraceae, Peptostreptococcaceae, Ruminococcaceae, and Ruminococcaceae. Not limited to these. Preferably, the bacterium present is from Clostridia taxa I, IV and / or XIVa. Preferably, the bacterium is Clostridia, which is often found in the lower gastrointestinal tract. For example, species detected in the lower gastrointestinal tract
Bacteria derived from the genus Clostridium (Classification Group 1), the preferred species for inclusion in the composition are Clostridium acetobutylicum, C. arbusti, Clostridium aurunchbutyricum (C). . aurantibutyricum), Clostridium beijerinckii, C. cellulovorans, C. cellulolyticum, C. thermocellum, C. thermobutyricum , C. pasteurianum, C. kluyveri, C. novyi, C. saccharobutylicum, C. thermosuccinogenes ), C. thermopalmarium, C. saccharolyticum, C. saccharoperbutylacetonicum, C. tyrobutyricum, Clostridium. C. tetanomorphum, C. magnum, C. ljungdahlii, C. autoethanogenum, C. butyricum, C. butyricum. puniceum), C. diolis, C. 5 homopropionicum and / or C. roseum;
Bacteria from the genera Christensenella, Eubacterium and Lacnospira (classification group XIVa), the preferred species for inclusion in the composition are Roseburia intestinalis, Roseburia bromii. ), Eubacterium rectale, Eubacterium hallii, Anaerostipes spp., Butyrivibrio spp. And / or Coprococcus spp. , But not limited to; and a bacterium from the genus Eubacterium (Class IV), the preferred species for inclusion in the composition includes, but is not limited to, Faecalibacterium prausnitzii. Not limited.

Preferably, the species in the composition is Clostridium butyricum.

Preferably, Clostridia is a butyrate-producing strain. Well-known Clostridium butyrate-producing strains include Anaerostipes, Butirivibrio, Coccoccus, Rosebria, Eubacterium-Lectare-related and Eubacterium-Harii-related species.

Preferably, the Clostridium species in the composition is capable of sporulation, preferably Clostridium butyricum. In a preferred embodiment, the strains are DSM10702 and ATCC19398.

The engineered bacterium may contain an unnatural gene capable of expressing (R) -3-HBD. Genes capable of expressing (R) -3-HBD (EC1.1.1.36) are Ralstonia eutropha, (Capriavidus necator), Bacillus sp, Klebsellia sp, It is selected from genes from organisms including, but not limited to, Pseudomonas sp, eg phbB and phaB.

Suitable genes, UniProt Accession Nos. P14697 (PHBB_CUPNH), P50203 (PHAB_ACISR), A0A060V147 (A0A060V147_KLESP), C1D6J5 (C1D6J5_LARHH), F8GXX8 (F8GXX8_CUPNN), F8GP10 (F8GP10_CUPNN), G0ETI7 (G0ETI7_CUPNN), A9LLG6 (A9LLG6_9BACI), A0A0E0VPS5 Includes (A0A0E0VPS5_STAA5), D5DZ99 (D5DZ99_BACMQ) and V6A8L4 (V6A8L4_PSEAI).

In certain embodiments, the (R) -3-HBD gene is phaB. The sequence of the phaB gene can be codon-optimized for the particular Clostridium species used. The sequence of phaB may include the sequence shown in FIG. 2 (SEQ ID NO: 1).

The nucleic acid encoding the unnatural (R) -3-HBD is a sequence having at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity with the phaB sequence (SEQ ID NO: 1) of FIG. May include.

Determining Identity Between Two Sequences There are numerous methods available. Preferred computer programs for determining identity between sequences include, but are not limited to, BLAST (Atschul et al, Journal of Molecular Biology, 215, 403-410, 1990). Preferably, the default parameters of the computer program are used.

In Clostridia, the natural enzyme catalyzes the 3-hydroxybutyrate reductase reaction. Therefore, in certain embodiments, the Clostridium species comprises a gene encoding an enzyme capable of converting (R) -3-hydroxybutyryl-CoA to (R) -3-HB.

Natural enzymes such as PTB and BUK convert (R) -3-hydroxybutyryl-CoA to (R) -3-HB via (R) -3-hydroxybutyrate-phosphate. Therefore, the genetically engineered bacterium may have a natural gene encoding PTB and BUK as well as an unnatural gene encoding (R) -3-HBD.

Clostridium species may also contain additional unnatural genes such as those encoding PTB, BUK, PCT and / or BUT.

Clostridium species may contain one or more unnatural genes encoding reductases capable of converting (R) -3-hydroxybutyryl-CoA to (R) -3-HB, such as ptb and buk. These genes can be derived from organisms including, but not limited to, Bacillus species, Escherichia coli or other species of Clostridia.

In addition, Clostridium species may contain one or more natural or unnatural genes encoding enzymes for producing SCFA, such as PCT or BUT. For example, PCT from Clostridium propionicum can be engineered into the strain to catalyze the CoA transfer reaction between (R / S) -3-hydroxybutyrate-CoA and acetate.

The term "non-natural gene" is a gene that is not in its natural environment and includes a gene from one species of microorganism that has been placed in another species of the same genus.

The unnatural gene can be codon-optimized for Clostridia and / or placed under the control of a promoter that allows controllable expression of the gene in Clostridia. The expression level of the enzyme can be optimized by controlling gene expression using inducible promoters and / or promoters of various intensities. In certain embodiments, the unnatural gene is controlled by a native Clostridia promoter, such as a ferredoxin or thiolase promoter. Other suitable promoters will be known to those of skill in the art.

Unnatural genes can be introduced into Clostridium strains by standard plasmid transformation techniques known in the field of recombinant microbial production, ie, by conjugation or electroporation. As an example, plasmid transformation is achieved by conjugation.

Unnatural genes, including (R) -3-HBD, can be applied to Clostridia chromosomes using gene integration technology known to those of skill in the art.

Clostridia are anaerobic bacteria that naturally undergo fermentative metabolism to convert carbohydrates into various reduced fermentation products. The bacterium has a unique metabolic pathway and biochemistry for the production of 3 and 4 carbon (C3 / C4) compounds.

The metabolic pathway of the genetically engineered Clostridium strain is shown in FIG. 1B. The genetically engineered Clostridium species carries a heterologous (R) -3-HBD (enzyme A in FIG. 1B) that converts acetoacetyl-CoA to (R) -3-hydroxybutyryl-CoA. The (R) specific 3-hydroxybutyryl-CoA dehydrogenase competes with the native HBD enzyme for the substrate (acetoacetyl-CoA). The native crotonase (Crt) enzyme has no or low activity on the (R) form of 3-hydroxybutyryl-CoA, where (R) -3-hydroxybutyryl-CoA is PTB and BUK or It enables conversion to (R) -3-HB by a natural enzyme such as BUT. The enzymes PTB and BUK are R morphologically specific and convert (R) -3-hydroxybutyryl-CoA to (R) -3-HB via (R) -3-hydroxybutyryl-phosphate.

The route used depends on the Clostridium species. In certain species commonly found in the Clostridium family (taxa I), including the genus Clostridium (including Clostridium butyricum), the final step requires two enzymes, PTB and BUK. In other species commonly found in the Lachnospiraceae family (taxon XIVa) and the Ruminococcus family (taxon IV), the final stage requires one enzyme, BUT. Certain Clostridia classes carry both enzymes and are capable of converting (R) -3-hydroxybutyryl-CoA to (R) -3-HB.

If PTB, BUK and / or BUT are not present in the native probiotic strain, the heterologous genes encoding these enzymes can be expressed in the engineered strain.

An alternative pathway to produce (R) -3-HB in genetically engineered anaerobic bacteria is, for example, by the introduction of a further unnatural gene encoding thioesterase, namely TesB from E. coli. These enzymes can convert (S)-and (R) -3-hydroxybutyryl-CoA to (S)-and (R) -3-HB, respectively.

Clostridium probiotics can be produced by fermentation performed under conditions suitable for the growth of the bacterium. After fermentation, the bacterium can be purified using centrifugation and prepared to retain activity. The bacterium in the composition is provided as a viable organism. The composition may contain bacterial spores and / or vegetative cells. The bacterium can be dried to retain the activity of the bacterium. Suitable drying methods include freeze-drying, spray-drying, heat-drying and combinations thereof. The resulting powder can then be mixed with one or more of the described pharmaceutically acceptable additives.

Spores and / or vegetative bacteria can be formulated with the conventional additives and ingredients for oral administration described herein. In particular, fat and / or aqueous ingredients, wetting agents, thickeners, preservatives, texturers, flavor enhancers and / or coatings, antioxidants, preservatives and / or pigments customary in the pharmaceutical and dietary supplement industry. .. Suitable pharmaceutically acceptable carriers include microcrystalline cellulose, cellobiose, mannitol, glucose, sucrose, lactose, polyvinylpyrrolidone, magnesium silicate, magnesium stearate and starch or combinations thereof. The bacterium can then be shaped into the appropriate orally ingestible form described herein. Suitable orally ingestible forms of probiotic bacteria can be produced by methods well known in the pharmaceutical industry.

In certain embodiments, an anaerobic bacterium producing 3-HB can be provided in a pharmaceutical composition in a wide range of concentrations as long as the bacterium is present in sufficient quantity to provide the desired therapeutic effect. Preferably, the bacterium is present in the pharmaceutical composition in an amount corresponding to 1 × 10 ⁵ to 1 × 10 ¹⁰ colony forming units / dry composition g (CFU / g), and more preferably the bacterium is. It is present in an amount corresponding to 1 × 10 ⁸ to 1 × 10 ¹⁰ CFU / dry composition g. If the composition is in the form of tablets, the bacterium may be present in an amount of 2 × 10 ^5-6 × 10 ⁷ CFU / tablet, preferably about 3 × 10 ^5-5 × 10 ⁷ CFU / tablet. Preferably, the bacterium grows and metabolizes in the colon and delivers about 1 μm to 10 mM, preferably about 100 μm to 5 mM, preferably about 1 mM 3-HB to the intestinal lumen.

"Delivery of 3-HB" means that 3-HB will be available at specific sites in the subject such that 3-HB exerts a therapeutic effect for treating an inflammatory disease, disorder or condition. means. 3-HB can be delivered to a particular site of inflammation in a subject such that 3-HB is available for having a local therapeutic effect. Preferably, the pharmaceutical composition of the present invention delivers 3-HB to the site of an inflammatory disease, disorder or condition and has a topical therapeutic effect. For example, 3-HB can be delivered rectal directly to the site of inflammation; 3-HB can be released at the site of inflammation from oral dosage forms such as capsules or tablets described herein; or 3-HB can be released at the site of inflammation. , 3-HB can be released from 3-HB delivery systems such as prodrugs or biological delivery systems that produce 3-HB at the site of inflammation.

The therapeutically effective amount of 3-HB to be administered depends on the 3-HB used (eg, (R) -isomer vs. (S) -isomer ratio), the subject to be treated, the type of severity and distress, and the method and route of administration. ..

Considering the amount of 3-HB delivered, the therapeutically effective amount is about 0.1 mg / kilogram (kg) body weight to about 500 mg / kg body weight, for example about 1 mg to about 250 mg / kg body weight, for example about 10 mg to about 180 mg. It can be / kg body weight, eg, about 20 mg to about 150 mg / kg body weight, eg, about 60 mg to about 125 mg / kg body weight. For example, a therapeutically effective amount can be from about 10 mg to about 40 g, such as from about 80 mg to about 20 g, such as from about 100 mg to about 15 g, such as from about 1 g to about 12 g, such as from about 5 g to about 10 g.

For oral administration, the therapeutically effective amount is about 10 mg to about 20 g, for example about 50 mg to about 20 g, for example about 100 mg to about 20 g, for example about 100 mg to about 10 g, for example about 500 mg to about 10 g, for example 500 mg to 5 g, for example 500 mg. ~ 2g, for example 1g ~ 15g, for example 1g ~ 10g, for example 1g ~ 8g, for example 1g ~ 2g, for example 1g ~ 4g, for example 2g ~ 4g, for example 2g ~ 6g, for example 4g ~ 8g, for example 4g ~ 6g, for example 5g It can be from 10 g, such as 6 g to 10 g, such as 6 g to 8 g, such as 8 g to 12 g, such as 8 g to 10 g, such as 10 g to 14 g, such as 10 g to 12 g, such as 10 g to 20 g. In a preferred embodiment, the therapeutically effective amount is about 10 mg to about 50 g, such as 10 mg to about 30 g, such as about 50 mg to about 30 g, such as about 100 mg to about 30 g, such as about 100 mg to about 15 g or, for example, about 500 mg to about 15 g. possible. In another preferred embodiment, the therapeutically effective amount is from about 1 g to 50 g, such as 5 g to 50 g, such as 10 g to 40 g, such as 1 g to 30 g, such as 5 g to 30 g, such as 3 g to 25 g, such as 1 g to 20 g, such as 5 g. It can be 20 g, eg 1 g-10 g, eg 20 g-30 g, eg 30 g-40 g or eg 5 g-15 g.

Each dose of the therapeutically effective amount can be a few unit dose. Single solid unit doses are, for example, about 50 mg to about 3 g, such as about 100 mg to about 2 g, such as about 250 mg to about 2 g, such as about 500 mg to about 2 g, such as about 250 mg to about 1 g, such as about 500 mg to about 1 g. It may contain, for example, 100 mg to 500 mg, such as 100 mg to 1 g, such as 100 mg to 2 g, such as 250 mg to 2 g, such as 250 mg to 1 g, such as 500 mg to 2 g, such as 500 mg to 1 g, such as 1 g to 3 g, such as 1 g to 2 g. Specific unit doses that may be noted are about 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g and 2 g, preferably about 1.8 g; or about 300 mg.

The dose amounts listed above may be given, for example, once, twice, three or four times a day or once or twice a week; preferably three times a day. For example, for oral administration, a total daily dose of 30 mg to about 120 g, such as about 240 mg to about 60 g, such as about 300 mg to about 45 g, such as about 3 g to about 36 g or, for example about 15 g to about 30 g, may be given. In certain preferred embodiments, the total daily dose for oral administration is, for example, about 1 g to about 50 g, such as about 1 g to about 30 g, such as about 5 g to about 30 g, such as about 5 g to about 25 g, such as about 5 g to about. It is 15 g, preferably about 5 g to about 10 g. Preferably, about 1.8 g is administered 3 times a day.

One dose can be administered as part of a meal or light meal or liquid, where the subject is provided with a dry dose to mix or combine with the meal, light meal or liquid (eg, water or fruit beverage).

INDUSTRIAL APPLICABILITY According to the present invention, 3-HB can be administered in combination with one or more additional therapeutic agents. Administration comprises administration of a formulation containing 3-HB and one or more additional therapeutic agents or essentially simultaneous, sequential or separate administration of separate formulations of 3-HB and one or more additional therapeutic agents. In certain embodiments, the 3-HB delivery means also delivers one or more additional therapeutic agents to the lumen of the gastrointestinal tract, preferably the additional therapeutic agent is butyrate.

The invention also includes methods of treating an inflammatory disease, disorder or condition in a subject.

Methods of treatment include amelioration of the disease, disorder or condition (ie, delaying, stopping or alleviating the onset of the disease, disorder or condition or at least one of its clinical manifestations); unrecognized by the patient. Reduction or improvement of at least one physical parameter; physical (eg, recognizable symptom stabilization), physiological (eg, physical parameter stabilization) or both physical, disorder or condition regulation; or disease Alternatively, it may be administered for the purpose of preventing or delaying the onset or onset of the disorder or its clinical symptoms.

A subject needs treatment if the subject benefits biologically, medically or quality of life from such treatment. Treatment is generally performed by a physician, who administers a therapeutically effective amount of 3-HB. Appropriately the subject is a human.

A therapeutically effective amount of 3-HB is an amount that is effective for the treatment, eg, delaying, stopping, alleviating or preventing the disease, disorder or condition or its symptoms. In general, subjects in need of treatment are those who exhibit symptoms of a disease, disorder or condition. Alternatively, the subject may be sensitive to the disease, disorder or condition, or test positive for the disease, disorder or condition, but may not be symptomatic.

Preferably, a therapeutically effective dose of 3-HB provides 3-HB at a concentration of about 1 μM to about 10 mM, preferably about 100 μM to about 5 mM, preferably about 1 mM into the lumen of the gastrointestinal tract, preferably the colon. do.

Inflammatory diseases, disorders or conditions specifically relate to inflammatory diseases, disorders or conditions, especially in the gastrointestinal tract including IBD and colorectal cancer such as Crohn's disease and ulcerative colitis. , High pro-inflammatory cytokines or proteins and / or may be characterized by inadequate anti-inflammatory cytokines or protein levels. Preferably, the inflammatory disease, disorder or condition is one or more of TNF-α, IL-23, IL-6, IL-1β, IL-12, MMP9 and NF-κB, preferably 2, 3, 4, 5 , 6 or more or all; preferably characterized by high levels of IL-23. Preferably, the inflammatory disease, disorder or condition is characterized by levels of IL-10 and / or TGF-β1 that are inadequate to achieve and maintain an appropriate immune response. Preferably, the inflammatory disease, disorder or condition is high levels of TNF-α, IL-23, IL-6, IL-1β, IL-12 and MMP9 2, 3, 4, 5 or higher or all. And due to inadequate levels of IL-10 and / or TGF-β1 to achieve and maintain an appropriate immune response; preferably of TNF-α, IL-23, IL-6, IL-1β, IL-12 and MMP9. It is characterized by high levels and inadequate levels of IL-10 and TGF-β1, and optionally further high levels of NF-κB.

Inflammatory diseases, disorders or conditions include IL-8, IL-18, IL-18 binding protein, IL-16, caspase-1, IL-1α, IL-17A and RANTES (CCL5), IL-22 and IL- It can be characterized by one or more high and / or inadequate levels of 27.

Inflammatory diseases, disorders or conditions include IBD such as Crohn's disease, ulcerative colitis, cystitis, collagenous colitis and lymphocytic colitis, colorectal cancer, rheumatoid arthritis, polysclerosis, psoriasis, psoriasis. It can be arthritis, gout, ankylosing spondylitis or COPD.

Preferably, the inflammatory disease, disorder or condition is an inflammatory disease, disorder or condition of the gastrointestinal tract, preferably the large intestine, more preferably the colon. Preferably, the inflammatory disease, disorder or condition is IBD, preferably Crohn's disease or ulcerative colitis or colorectal cancer.

The invention also comprises compositions comprising biological delivery systems such as genetically engineered anaerobic bacteria capable of producing (R) -3-HB described herein and in the treatment of gastrointestinal diseases and disorders and in animals. Regarding its use as a probiotic for health.

The bacteria are conventional additives and ingredients for such oral compositions, ie fat and / or aqueous ingredients, wetting agents, thickeners, preservatives, texturers commonly used in the pharmaceutical and dietary supplement industry. , Flavor enhancers and / or coatings, antioxidants, preservatives and / or pigments. Suitable pharmaceutically acceptable carriers include microcrystalline cellulose, mannitol, glucose, polyvinylpyrrolidone and starch or combinations thereof. The bacterium can then be shaped into a suitable orally ingestible form. Suitable orally ingestible forms of probiotic bacteria can be produced by methods well known in the pharmaceutical industry.

Compositions administered orally are in the form of coated tablets, gel capsules, gels, emulsions, tablets, capsules, hydrogels, food bars, compressed or loose powders, liquid suspensions or solutions, confectionery products or food products. Can be manufactured. Preferably, the composition is in dry form. The preferred oral form for the composition is a solid form such as a capsule, tablet or powder.

The composition can be formulated by conventional methods for the production of oral formulations, in particular coated tablets, gel capsules, gels, emulsions, tablets, capsules, hydrogels and powders.

Orally ingestible carriers can also be food products such as beverages, drinks, dietary supplements or functional foods.

The genetically engineered anaerobic bacteria that produce (R) -3-HB can also be incorporated as part of a food product, i.e. in yogurt, milk or soy milk, or as a dietary supplement. Such foods and dietary supplements can be produced by methods well known in the food and dietary supplement industries.
The composition can be incorporated into animal feeding products as a food additive.

The composition can also be used for the prevention or treatment of bacterial flora abnormalities. Gastrointestinal flora abnormalities can be caused by the use of broad-spectrum antibiotics. The composition can be used for the treatment and prevention of bacterial flora abnormalities by administration of antibiotics.

During bacterial flora abnormalities, subjects are susceptible to opportunistic pathogenic microorganisms, including Clostridium difficile. The composition can be used for the treatment or prevention of bacterial infections. In certain embodiments of the invention, the composition can be used to treat or prevent Clostridium difficile infection.

Compositions comprising genetically engineered anaerobic bacteria that produce (R) -3-HB can also be used to regulate the gut flora in a subject. A composition comprising or will be essentially a genetically engineered anaerobic bacterium that produces (R) -3-HB is administered to a healthy subject, i.e., for non-therapeutic use. The composition can be orally administered to the subject.

The degree of growth and colonization of the genetically engineered bacterium in the gut is prebiotic with species and strain selection and / or the particular food in which the bacterium propagates, within the same dose as containing the probiotic or separately. It can be controlled by providing it as a composition to be ingested into.

The composition may also further comprise a prebiotic to enhance the growth of the administered probiotic. The prebiotic can be administered sequentially, simultaneously or separately with a composition comprising a genetically engineered anaerobic bacterium that produces (R) -3-HB. Prebiotic and genetically engineered bacteria can be formulated together in the same composition for co-administration. Alternatively, the bacterium and prebiotic may be formulated separately for simultaneous or sequential administration.

Prebiotics are substances that promote the growth of probiotics in the intestinal tract. They are food substances that are fermented in the intestinal tract by the bacteria. The addition of prebiotics provides a medium that can promote the growth of probiotic strains in the intestinal tract. One or more monosaccharides, oligosaccharides, polysaccharides or other prebiotics that promote bacterial growth may be used.

Preferably, the prebiotic can be selected from the group consisting of oligosaccharides comprising fructose, galactose, mannose; dietary fiber, in particular soluble fiber, soybean fiber; inulin; or a combination thereof, if desired. Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalt-oligosaccharides, xylooligosaccharides, soybean oligosaccharides, glycosyl sucrose (GS), lactosucrose (LS), lactulose. (LA), palatinose-oligosaccharides (PAOs), malto-oligosaccharides, pectins, hydrolyzates thereof or combinations thereof are selected from the group.

(R) -3-HB secreted by the bacterium acts locally within the lumen and / or mucosal layer of the intestine, eg, by acting as an enhancer and / or suppressor of microbial growth, intestinal bacteria. It can regulate the flora.

All of the properties described herein (including the appended claims, abstracts and drawings) and / or all of the steps of any disclosed method, at least a portion of such properties and / or steps. Any combination can be combined with any of the above embodiments, except for combinations that are mutually exclusive. In particular, any of the activators and compositions described herein can be used in any of the treatment methods described. It is clearly contemplated that any and all such combinations form part of the present invention.

The present invention will then be described in more detail with reference to the following examples.

Example 1-Production of (R) -3-HB in Clostridium butyricum expressing phaB 1) Gene synthesis The gene Cupriavidus necator phaB was codon-optimized for Clostridia. FIG. 2 shows an example of a codon-optimized sequence synthesized by Gene Art® (Thermo Fisher Scientific).

2) plasmid construction phaB was cloned into the plasmid pMTL83251 under the control of the Clostridium sporogenes Pfdx promoter using standard cloning techniques to obtain the plasmid pMTL83251_pfdx_phaB (Fig. 3).

3) Strain preparation plasmid pMTL83251_pfdx_phaB was conjugated to Clostridium butyricum ATCC19398 / DSM10702 using Escherichia coli CA434 as a conjugation donor. A strain-specific conjugation protocol was applied. Briefly, overnight cultures of E. coli and Clostridium butyricum CA434 carrying the plasmid pMTL83251_pfdx_phaB were inoculated into 9 ml LB and RC broth, respectively. Cultures were grown until OD ₆₀₀ reached 0.5-0.7. 1 ml of E. coli culture was translocated and the pellet was mixed with 200 μl heat shock (50 ° C., 10 min) Clostridium butyricum culture. The cell mixture was spotted on non-selective RCM plates and incubated overnight. The incubation mixture was resuspended in 500 μl fresh RCM and seeded in selective medium containing 10 μg / ml erythromycin. The presence of the plasmid in the resulting conjugation completed was confirmed by PCR using plasmid-specific primers.

4) Fermentation data on Clostridium butyricum
Proliferation method
RC broth containing 13 g of yeast extract, 10 g of peptone, 1 g of soluble starch, 5.0 g of sodium chloride, 3 g of sodium acetate, 0.5 g of cysteine hydrochloride and 2% of carbohydrate was used per liter. Calcium carbonate 10 g / L was added to the liquid culture for pH control. The solid medium contained 15 g / L agar.
Transformants were grown overnight in seed culture (RC broth) at 37 ° C. 100 ml RC broth containing 2% glucose was inoculated from 0.05 to 0.1 starting OD. The strain was anaerobicly grown at 37 ° C. in the presence of the required antibiotics. Samples for metabolic analysis were taken at regular intervals.

Analysis and Results The engineered Clostridium butyricum (CHN-1) culture supernatant was analyzed for (R) -3-HB and (S) -3-HB using the 3-HB assay kit (Sigma Aldrich). did. The strain expressing phaB produced only (R) -3-HB. Culture supernatants of CHN-1 and native Clostridium butyricum were also analyzed for SCFAs and (R) -3-HB production using HPLC-RI. The phaB-expressing strain of Clostridium butyricum (CHN-1) was produced at about 187 mg / L after 24-hour growth, as shown in FIG. 4B. The wild-type Clostridium butyricum strain produced only butyrate and acetate, as shown in FIG. 4A.

Example 2-Preparation for colon delivery
Zacol NMX® is a dietary supplement (functional food) for the colon based on MMX® technology. It is a product based on the provision of MMX® technology that combines the calcium salt of butyric acid with inulin. NMX® is a functional food version of MMX® technology. The tablets are calcium 3-HB (0.307 g), maltodextrin, inulin (0.250 g), sorbitol, hypromellose, microcrystalline cellulose, processed corn starch, citric acid, colloidal silica hydrate, talc, shelac, stearic acid. Magnesium, stearic acid, lecithin, titanium dioxide, hydroxypropyl, triethylcitrate; aroma: contains vanillin.
BioCare format. Capsules contain 1815 mg 3-HB, 243 mg calcium hydroxide, 123 mg magnesium hydroxide, medium chain triglycerides, capsule shells (hydroxypropylmethylcellulose), anticaking agents (silicon dioxide and magnesium stearate). Take 1 capsule 3 times daily after meals or as directed by a specialist.

Example 3-Bacterial Delivery The same culture medium and inoculation technique as for sporulation fermentation of Clostridium butyricum was used. Samples were taken at regular intervals at the start of the experiment and over 72 hours to determine the ratio of vegetative cells to spores. For spore counting, the sample was heat treated at 65 ° C. for 30 minutes to kill all vegetative cells. At the same time, samples taken for total CFU count (nutrient cells + spores) were placed on the bench to prevent further growth in the medium. Heat-treated and non-heat-treated samples were then serially diluted and seeded in 20 μL individual spots in triplicate on non-selective medium for wild-type and selective medium for engineered strains. After overnight incubation at 37 ° C., CFU / mL was determined. 5A-B show the development of spores over 72 hours. FIG. 6 shows the percentage of spores in total CFU in culture over 72 hours.
The tablet formulation contains cornstarch, lactose, hydrated magnesium silicate, microcrystalline cellulose, magnesium stearate and sucrose.

Example 4-Evaluation of CHN-1 in Simulated Colon Environment Spores of engineered Clostridium butyricum (CHN-1) were produced in a pH controlled laboratory scale bioreactor. Strains were treated at 37 ° C. on nitrogen and carbon dioxide flash anaerobic workstations prior to inoculation to the bioreactor.
CHN-1 was grown from spore stock on fortified Clostridium agar (Sigma-Aldrich, UK) plates. Single colonies for ingestion of modified fortified crostridium (RC) broth (13 g yeast, 10 g peptone, 1 g starch, 5 g NaCl, ₃ g CH ₃ COONa, 0.5 g cysteine hydrochloride, 10 g CaCO, 20 g glucose per liter). It was used and then it was ^{serially diluted 100-10-8 with} modified RC broth. After 8-12 hours, 1:10 dilutions were prepared in freshly modified RC from maximum dilution overnight culture growth (usually ^10-6 ) for daily cultures. The 1-day cultures were generally grown for 1 1/2 to 2 hours prior to transfer to serum bottles. The serum bottle was covered with a rubber stopper to maintain anaerobic growth. Bacterial cultures from serum bottles were used and ingested into the bioreactor at 1:10. The bioreactor containing the modified RC was pH controlled to 6.5 with 3M sterile KOH with 125 rpm stirring and 6 L / hour N ₂ flushing as needed. The bioreactor was maintained at 37 ° C. for a long time. Cell masses were harvested 24 hours later, stored at 4 ° C. and then purified. The vegetative cells were destroyed by heat treatment at 65 ° C. Purification involved a repetitive wash step using sterile deionized water and centrifugation at 5000 xg for 20 minutes. Spores were counted using an improved Neubawell blood cell counter and the number of surviving spores was assessed by colony forming units on RC agar.

The ability of CHN-1 spores to germinate and proliferate in a colonic environment was evaluated using the proximal colon simulation conditions described in Molly et al., (1993) Appl Microbiol Biotechnol. Reducing sugar pre-depleted basal colon medium containing nutrients present in the colon (eg host or dietary glycans such as mucin or starch) was added to the double jacket glass bioreactor. CHN-1 spores and / or faecal inoculum were added to the bioreactor in an anaerobic workstation. The stool inoculum material was prepared by mixing the stool donor material of one healthy donor with a fresh stool sample and pre-reduced phosphate buffer at a ratio of 1: 5 and removing the particles by centrifugation at 500 × g. The inoculum was then added to the bioreactor at a 1:10 dilution. The bioreactor was sealed with a rubber stopper to maintain anaerobic growth.
The experiment included four different conditions in a triplet: i) inoculated with filtered sterile fecal suspension and CHN-1; ii) inoculated with filtered sterile fecal suspension, CHN-1 and glucose (1 g / L); iii) Inoculate stool suspension and CHN-1; and iv) Inoculate stool suspension. The bioreactor was maintained at 37 ° C. and continuous mixing at 90 rpm was applied. Samples were taken for analysis at t = 0 hours, 2 hours, 4 hours, 6 hours, 24 hours, 30 hours and 48 hours after inoculation.
Spore germination, proliferation and CHN-1 metabolic activity were assessed by 1) colony forming units on selective medium; 2) pH reduction; 3) SCFA production; and 4) (R) -3-HB production. 5) CHN-1 detection was performed using two specific PCR protocols for the detection of the Clostridium butyricum 16s-23s intergenic spacer region and the detection of phaB.

1) Colony forming units were evaluated using modified Clostridium butyricum isolated medium (BIM) as described in Popoff (1984) J Clin Microbiol, using D-cycloserine as the only antibiotic at a concentration of 250 μg / mL. This medium allows for selective enumeration of CHN-1 and no colonies are observed in the background without supplementation of this strain (detection threshold 200 CFU / mL). Total viable cell count (FIG. 7) was assessed by serially diluting the sample and seeding in modified BIM. Colony forming units were counted after overnight incubation in anaerobic conditions. The number of heat-resistant cells (FIG. 8) was evaluated by pasteurizing the sample for 30 minutes at 65 ° C., serially diluting and seeding in modified BIM. Colony forming units were counted after overnight incubation under anaerobic conditions.
As shown in FIG. 7, there was no significant difference in the number of viable cells counted in each of the reactors inoculated with the filtered sterile fecal suspension and CHN-1 with or without glucose. In a bioreactor inoculated with CHN-1 and fecal suspension, there was a constant increase in viable cell count for the first 6 hours of the experiment, indicating germination and proliferation of CHN-1. After 24 hours, the number of viable cells of CHN-1 was statistically significantly higher in the bioreactor inoculated with the filtration-sterilized fecal suspension compared with the inoculated one (p <0. 05), it was shown that there was competition between the fecal flora and CHN-1 in these bioreactors. Bioreactors not inoculated with CHN-1 did not recover CFU above the detection threshold.
As shown in FIG. 8, there was no significant difference in the thermal resistance counts counted in the respective bioreactors inoculated with the filtered sterile fecal suspension and CHN-1 with or without glucose. There was a constant decrease in thermal resistance counts in all bioreactors at 4-24 hours of incubation, indicating germination and elongation from spores leading to vegetative cell proliferation.

2) The decrease in pH caused by metabolic activity in the simulated colon (Fig. 9) was measured at t = 0 hours, 6 hours, 24 hours and 48 hours using the Senseline F410 pH meter (ProSense, Oosterhout, NL). did.
As shown in FIG. 9, there was no significant difference in pH between the four experimental settings at the start of the experiment. The pH of the filtered sterile stool inoculum and the bioreactor inoculated with CHN-1 and the stool suspension and the bioreactor inoculated with CHN-1 were also not significantly different at any time during the experiment. However, the pH of the filtered sterile stool suspension and the bioreactor inoculated with CHN-1 was statistically significantly different from that with glucose added after 6 hours (p <0). .02). Bioreactors infused with fecal suspension also had statistically significant differences at 6 and 24 hours compared to those with and without CHN-1 (p =, respectively). 0.003 and 0.0133). This indicates that CHN-1 is metabolically active in the filtered sterile stool and stool suspensions, respectively, in the background within the first 6 hours of instillation.

3) Production of SCFA acetate (Fig. 10) and butyrate (Fig. 11) was measured by gas chromatography as described in De Weirdt et al. (2010) FEMS Microbiol Ecol.
As shown in FIG. 10, when the fecal inoculation material and the bioreactor supplemented with CHN-1 were compared with the fecal inoculation material alone, there was a statistically significant increase in acetate concentration from 6 hours after incubation to the end of the experiment. (P <0.04). Significantly higher amounts of acetate were present after 24 hours in bioreactors containing stool inoculum and CHN-1 compared to those containing filtered sterile stool suspensions and CHN-1 (p < 0.003).
As shown in FIG. 11, in the bioreactor supplemented with the fecal inoculation material and CHN-1, a statistically significant increase in the butyrate concentration was observed throughout the experiment as compared with the bioreactor supplemented with the fecal suspension alone. (P = 0.0001). There was no significant difference between bioreactors containing CHN-1 with or without fecal suspension. Highest amounts of butyrate were found in reactors supplemented with glucose as a precursor for butyrate production (p <0.04).

4) Aminex with a particle size of 9 μm at 300 mm × 7.8 mm on the Dionex UltiMate 3000 System (Thermo Scientific, USA) with (R) -3-HB production in the simulated colon set at 35 ° C. and 55 minute runtimes. Measured by HPLC-RI using HPX-87H column (Biorad, USA). As the mobile phase, 5 mM H ₂ SO ₄ was used at a flow rate of 0.5 mL / min. Culture samples were filtered and sterilized prior to analysis using a 13 mm diameter KX syringe filter (Kinesis Ltd, UK) of regular cellulose with a pore size of 0.22 μm. A known amount of (R) -3-HB was added to the sample and then the sample was mixed 1: 1 with a mobile phase containing 50 mM valerate as an internal standard. A calibration standard containing increasing concentrations of glucose, (R) -3-HB, butyrate, acetate and lactate was run simultaneously with the added sample.
As shown in FIG. 12A, a significantly higher amount of (R) -3-HB was produced in the filtered sterile stool suspension, CHN-1 and glucose inoculated bioreactor compared to the glucose-free bioreactor. rice field. In any of the bioreactors supplemented with CHN-1, a significantly higher amount of (R) -3-HB was present (p <0.03) as compared to the bioreactor supplemented with only the fecal suspension. This indicates that the production of (R) -3-HB in all bioreactors depends on the presence of CHN-1.
FIG. 12B shows the (R) -3-HB concentration measured at each of the three experimental iterations performed for the third experimental condition of FIG. 12A (inoculated with fecal suspension and CHN-1). These three values are combined and shown in FIG. 12A. (R) -3-HB was detected in all three iterations above the baseline concentration found in human serum (0-200 μM) under feeding conditions. One reactor showed a level of about 1 mM. This range of 200 μM to 1 mM (R) -3-HB is described in Examples 5 and 6 in two human colon tissue-based in vitro models with reduced expression of multiple inflammatory proteins and anti-inflammatory proteins. It was found to be effective in increasing expression.

ゲノムＤＮＡを、フェノール－クロロホルム抽出した後、両オリゴヌクレオチドセットを使用するＰＣＲに付した。 5) The presence of CHN-1 in the bioreactor was further confirmed by strain-specific PCR. For this, two different sets of oligonucleotides were used (Table 1). The first set amplified the 16s-23s intergenic spacer region of Clostridium butyricum as described in Nakanishi et al. (2005) Microbiol Immunol. The second set specifically amplified the integrated phaB gene.

Genomic DNA was extracted with phenol-chloroform and then subjected to PCR using both oligonucleotide sets.

As shown in FIG. 13, the presence of Clostridium butyricum was confirmed in Samples 1-9 corresponding to the bioreactor supplemented with CHN-1 spores. Low levels of Clostridium butyricum (below viable cell count and thermal resistance count detection levels (FIGS. 7 and 8)) were also confirmed in reactors 10-12 to which only the fecal suspension was added.
As shown in FIG. 14, the presence of phaB was confirmed in samples 1-9 corresponding to the bioreactor supplemented with CHN-1 spores. Amplicons were not observed in reactors supplemented with fecal suspension only, confirming the absence of phaB in these bioreactors.
Taken together, these data indicate that CHN-1 spores can germinate in a simulated colonic environment. Germination of these spores subsequently leads to vegetative cell proliferation, which is now indicated by indicators of metabolic activity, which is SCFA concentration and pH reduction. The data also show that CHN-1 succeeded in introducing a novel metabolite, (R) -3-HB, which is not normally found in the colonic environment.

Example 5-Modeled Inflammation Using Primary Human Intestinal Organoids To measure the effect of (R) -3-HB on inflammation of primary human intestinal organoids, a healthy colon sample of a 67-year-old female donor was sampled 8 before the experiment. In vitro cultured for weeks. The method used in Hannan et al., Stem Cell Reports. Vol. 1, 293-306 October 15, 2013.

1) Organoids to induce inflammation, TNFα (40 ng / mL) alone or in combination with butyrate (10 μM), (R) -3-HB (10 μM) or butyrate and (R) -3-HB for 18 hours Processed. The cells were subsequently lysed and RNA was isolated for cDNA synthesis and measurement of mRNA expression levels of inflammatory factors, NF-κB and TNFα by qPCR.
FIG. 15 responds to incubation with butyrate, (R) -3-HB or TNFα in combination with butyrate and (R) -3-HB, normalized to a control (non-stimulated sample) set to 1. The relative mRNA expression levels of the inflammatory factors NF-κB (A) and TNFα (B) expressed by the primary human intestinal organoids are shown. The mRNA expression of the inflammatory factors NF-κB and TNF-α was reduced by treatment with butyrate, (R) -3-HB or a combination of butyrate and (R) -3-HB.

2) Organoids were treated with TNF-α (40 ng / mL) alone or with (R) -3-HB (sodium salt 10 μM) for 18 hours to induce inflammation. Subsequently, cells were lysed, RNA was isolated for cDNA synthesis, and mRNA expression levels of a group of inflammatory factors were measured by qPCR.
FIG. 16 is expressed by primary human intestinal organoids in response to incubation with TNF-α alone or in combination with (R) -3-HB, normalized to a normalized control (non-stimulated sample set to 0). The relative mRNA expression level of the inflammatory factor was shown. MRNA expression of pro-inflammatory cytokines and proteins IL-23, TNF-α, IL-1β, IL-6 and NF-κβ is present in (R) -3-HB compared to treatment with TNF-α alone. Reduced below. MRNA expression of the anti-inflammatory cytokines TGF-β1 and IL-10 was increased in the presence of (R) -3-HB compared to treatment with TNF-α alone.
Both butyrate and (R) -3-HB act on pro-inflammatory cytokines and proteins as well as anti-inflammatory cytokines and proteins. (R) -3-HB has a greater reducing effect than butyrate on several important pro-inflammatory regulators of IBD and a greater provocative effect than butyrate on the major protective regulator of intestinal inflammation (R). No data shown).

3) The effects of (R) -3-HB and related SCFA butyrate on the inflammatory response caused by primary human intestinal organoids by co-incubation with TNF-α as an inflammatory stimulus were measured. Primary human intestinal organoids were cultured for 8 weeks prior to the experiment as described in Hannan et al. (2013) Stem Cell Reports.
To induce inflammation, organoids were treated with 60 ng / mL TNF-α alone or with various concentrations of (R) -3-HB sodium salts or butyrate for 24 hours. The cells were subsequently lysed and RNA was isolated using the RNeasy mini kit (Qiagen Ltd, Germany) for cDNA synthesis. The mRNA expression level of IL-23 was measured by qPCR using the SensiMix SYBR low-ROX kit (Bioline, UK).
Values were normalized to untreated controls and graphs show differences in mRNA levels measured with TNF-α alone (set as 0) and with organoids treated with butyrate or (R) -3-HB with TNF-α. Is shown.
FIG. 17 shows the relative mRNA expression levels of IL-23 in organoids treated with 60 ng / mL TNF-α and increasing concentrations of butyrate or (R) -3-HB. These data indicate that a concentration of (R) -3HB at a concentration of 10-100 μM may be effective in reducing IL-23 expression in the human intestinal mucosa. IL-23 is an important mediator of inflammation in IBD and is the target of several pharmaceutical monoclonal antibody drugs. This concentration range of (R) -3-HB is achievable in the intestinal lumen using bacterial delivery, as evidenced by in vitro intestinal modeling (see Figure 12). (R) -3-HB has a greater IL-23 expression-reducing effect than butyrate at these concentrations.

Example 6-Modeled Inflammatory Colon Rectal Specimens Using the Exobibohuman Colon Tissue Model can be obtained by surgical resection or histology of intestinal tissue after obtaining the patient's informed consent document.

Materials: Specimen collection pot, sterile scalpel, sterile forceps, Petri dish, PBS, Dulbecco's minimum essential medium (DMEM) high glucose, fetal bovine serum (FCS), L-glutamine, antibiotics, 96-well U-bottom sterile plate with lid, Multi-channel pipetter.

Preparation of extracolonic and rectal implants. After tissue resection, the specimen is maintained in a specimen collection pot containing DMEM for transport to the laboratory. Transfer the specimen to a Petri dish containing some medium. Remove the muscle from the resected specimen with a sterile scalpel. Transfer the remaining tissue to a new Petri dish containing medium and cut the tissue into explants containing both epithelial mucosa and muscularis mucosae (Berry N, Herrera C, Cranage M. Methods Mol. Biol. 2011; 665: 133-60). Proceed to the assay.

Preclinical evaluation of candidate compounds. To induce inflammation, explants are incubated with 100 μl inflammatory stimuli (TNF-α 15 μg / ml) in 96-well U-bottom sterile plates. The explants are incubated with 100 ml of complete medium (FCS, L-glutamine and DMEM containing antibiotics) for 1 hour. To assess the activity of the compound of interest, 100 μl of compound (10 mM) is added to the explants. The tissue explants are then cultured at 37 ° C. for 24 hours in an atmosphere containing 5% CO ₂ . Negative controls are tissue explants cultured without the addition of any inflammatory irritation or target compound (200 μl complete medium addition). Positive controls are explants cultured only with inflammatory stimuli. After culturing, the tissue explants are removed from the culture wells, the plates are settled, and 180 μl of the culture supernatant is collected. Freeze the supernatant and tissue explants at -80 ° C. The supernatant and / or tissue explants are then quantified.

The Luminex method was used to determine the protein concentration in the culture supernatant:
Luminex Protocol: The in-house assay was performed by Dr. Carolina Herrera of St Mary's Hospital, Paddington, London. Luminex assay buffer: PBS, goat serum, mouse serum, Tween 20, Tris pH 7-8. Luminex wash buffer: PBS, Tween-20. operation:
Assay plate preparation 1. Assay plate is pre-humidified with assay buffer Preparation of sample 1. Preparation of 11 sample at 1/3 dilution stage Sample preparation 1. Sample diluted bead preparation as needed for each panel 1. Super beads Sonic and vortex treatment 2. Dilute the beads with assay buffer Assay setup 1. Remove assay buffer from the plate 2. Add 50 μl of specimen, blank and sample to the corresponding wells 3. Add 50 μl of bead mixture to each well 4. Cover the plate with foil and shake with a plate shaker for 1 hour and 30 minutes.

Addition of detection antibody cocktail 1. Dilute detection antibody cocktail with assay buffer 2. Wash plate 3 times with wash buffer 3. Add 50 μl detection antibody cocktail per well 4. Cover with foil as described above and shake for 1 hour Streptavidin -PE addition 1. Streptavidin-PE diluted with assay buffer 2. Plate washed 3 times with buffer 3. 50 μl diluted Streptavidin-PE added to each well 4. Cover with foil and shake for 30 minutes 5 .Wash 3 times and add 100 μl of sheath solution to each well. 6. Shake the plate with Luminex before running it or store in a refrigerator and coat with foil until the next day.

FIG. 18 shows incubation with TNF-α alone (60 minutes) or TNF-α (60 minutes) normalized to a normalized control (non-stimulated sample set to 0), followed by (R) -3-. The relative protein levels of the inflammatory factor expressed by the Exvivo colon tissue sample in response to exposure to HB (24 hours) are shown. Both sodium salts and free acids of 3-hydroxybutyric acid were all tested at 10 mM concentrations. Relative protein levels [ng / mL] were determined using a standard curve. Protein concentrations of pro-inflammatory cytokines and proteins IL-12, IL-1β and IL-6 were reduced in the presence of (R) -3-HB compared to treatment with TNF-α alone. The protein concentration of the anti-inflammatory cytokine IL-10 was increased in the presence of (R) -3-HB compared to treatment with TNF-α alone. Both butyrate and (R) -3-HB act on pro-inflammatory cytokines and proteins and anti-inflammatory cytokines and proteins. (R) -3-HB has a greater reducing effect than butyrate on several important pro-inflammatory regulators of IBD and a greater provocative effect than butyrate on the major protective regulators of intestinal inflammation (R). No data shown).

The Proteome Profiler ^TM Array was used to measure the relative abundance of large sets of proteins and cytokines involved in the immune response. The experimental conditions and colon tissue sample preparation were the same as above. The Human XL Cytokine Array Kit (www.rndsystems.com), Catalog Number ARY022, was used for this experiment. Cytokines, chemokines and growth factors are extracellular signaling molecules that mediate intercellular communication. These molecules are released from cells and have important roles in many biological processes such as cell proliferation, differentiation, gene expression, migration, immunity and inflammation. In most biological processes, multiple cytokines operate in large networks, where the action of one cytokine is regulated by the presence or absence of another cytokine. The Human XL Cytokine Array Kit is a fast, sensitive and economical tool for simultaneously detecting cytokine differences between samples. The relative expression levels of 102 human soluble proteins can be determined without performing a number of immunoassays.

Assay Principles Capture and control antibodies are duplicated and spotted on a nitrocellulose membrane. Dilute cell culture supernatant, cell lysate, serum, plasma, human milk, urine, saliva or tissue lysate and incubate overnight with Proteome Profiler Human XL Cytokine Array. The membrane is washed to remove unbound material, followed by incubation with a cocktail of biotinylated detection antibody. Streptavidin-HRP and chemiluminescence detection reagents are then applied and a signal corresponding to the amount of bound protein is generated at each capture spot.
FIG. 19 shows incubation of TNF-α alone (60 minutes) or TNF-α (60 minutes) normalized to a normalized control (non-stimulated sample set to 0), followed by (R) -3-HB. The relative protein abundance (non-quantitative) of the inflammatory factor expressed by the Exvivo colon tissue sample in response to exposure to (24 hours) is shown. Both sodium salts and free acids of 3-hydroxybutyric acid were all tested at 10 mM concentrations. Protein concentrations of pro-inflammatory cytokines and proteins TNF-α, IL-23 and MMP9 were reduced in the presence of (R) -3-HB compared to treatment with TNF-α alone.

Example 7-Germination and Elongation Competitive Assay Clostridium difficile and genetically engineered Clostridium butyricum spores were obtained by harvesting spore stock from a nutrient-rich medium plate after 5 days incubation. The sample was heat treated at 65 ° C. for 30 minutes to kill any remaining vegetative cells. For spore germination, 50 μL of spore culture was inoculated into 10 mL of nutrient medium supplemented with 0.1% sodium taurocorate (Clostridium difficile germination inducer). Clostridium difficile and Clostridium butyricum spores were used in single and co-cultured triplet experiments. After incubation at 37 ° C. for 24 hours under anaerobic conditions, samples were taken from each individual experiment, serially diluted and inoculated into 20 μL individual spots in triplicate on eutrophic medium. After overnight incubation at 37 ° C., CFU / mL was determined.
FIG. 20 shows CFU / mL obtained by single or co-culture of Clostridium difficile and Clostridium butyricum spores. Germination and / or elongation of Clostridium difficile was completely blocked when spores were germinated by co-culture alongside Clostridium difficile spores.

Example 8-Survival assessment under gastric and small intestinal conditions CHN-1 spores were resuspended in 1 mL PBS and g / L arabinogalactan 1, pectin 2, xylan 1, starch 3, glucose, 0. It was inoculated into 9 mL of simulated gastric medium (GSM) containing 4, yeast extract, 3, pectin, 1, mucin, 4, cysteine, 0.5 and pepsin 1. Medium was adjusted to pH 3 using 1M HCl prior to autoclaving. GSM cultures are anaerobically incubated for 2 hours at 37 ° C. with stirring at 100 rpm, then 5 mL pre-preparation containing pancreatine, 3, dehydrated bile extract, 8, sodium bicarbonate, 10 at g / L. Reduced pancreatic bile was added. The cultures were anaerobicly incubated for 4 hours at 37 ° C. with stirring at 50 rpm. GSM was not pre-reduced, but was stored at 37 ° C. prior to transfer to cabinet to mimic the aerobic conditions encountered in the stomach and the oxygen loss encountered due to gastrointestinal migration due to subsequent anaerobic incubation. Samples were taken at t = 0 hours, 2 hours, 4 hours and 6 hours of incubation. The sample was serially diluted and three separate 20 μL spots were seeded on the RCM agar plate. Plates were anaerobically incubated overnight at 37 ° C. and colonies were counted to assess spore viability under gastric and small intestinal conditions.
FIG. 21 shows that CHN-1 spores survive gastric acid conditions and are therefore viable.
Furthermore, the present invention includes the following aspects.
Item 1
3-Hydroxybutyric acid (3-HB) or a salt thereof for use in a method of treating an inflammatory disease, disorder or condition in a subject.
Item 2
Item 3. The 3-HB according to Item 1, wherein at least about 90% of 3-HB is (R) -isomer ((R) -3-HB).
Item 3
A pharmaceutical composition comprising 3-HB or a salt thereof for use in a method of treating an inflammatory disease, disorder or condition in a subject.
Item 4
Item 3. The pharmaceutical composition for use according to Item 3, wherein the composition is formulated to deliver 3-HB or a salt thereof to the gastrointestinal tract, preferably the lumen of the gastrointestinal tract.
Item 5
A pharmaceutical composition for use in a method of treating an inflammatory disease, disorder or condition in a subject, wherein the composition comprises a 3-HB delivery means, wherein the method is a 3-HB delivery means of the gastrointestinal tract. A pharmaceutical composition comprising delivery to the lumen.
Item 6
Item 5. The pharmaceutical composition for use according to Item 5, wherein the 3-HB delivery means is a biological delivery system for delivering a prodrug or 3-HB to the lumen of the gastrointestinal tract.
Item 7
Item 6. The pharmaceutical composition for use according to Item 6, wherein the biological delivery system comprises an anaerobic bacterium that produces 3-HB.
Item 8
Item 5. The pharmaceutical composition for use according to Item 7, wherein the anaerobic bacterium is genetically engineered.
Item 9
Item 8. The pharmaceutical composition for use according to Item 8, wherein the anaerobic bacterium is a butyrate-producing bacterium containing an unnatural gene capable of expressing (R) -3-hydroxybutyryl-CoA dehydrogenase.
Item 10
Item 9. The pharmaceutical composition for use according to Item 9, wherein the anaerobic bacterium is an obligate anaerobic bacterium, preferably a spore forming Clostridium butyricum.
Item 11
Item 6. The pharmaceutical composition for use according to any one of Items 3 to 10, wherein at least about 90% of 3-HB is (R) -isomer ((R) -3-HB).
Item 12
Item 6. The pharmaceutical composition for use according to any one of Items 3 to 11, wherein the pharmaceutical composition is formulated for release control.
Item 13
Item 6. The pharmaceutical composition for use according to any one of Items 5 to 12, wherein the pharmaceutical composition is for oral administration.
Item 14
Item 13. The pharmaceutical composition for use according to Item 13, wherein the pharmaceutical composition comprises a release control layer or coating that surrounds a core comprising 3-HB or a salt thereof or a means of delivery.
Item 15
13. The pharmaceutical composition for use according to item 13 or item 14, wherein the pharmaceutical composition is formulated to deliver 3-HB or a salt thereof or a means of delivery to the gastrointestinal tract at a pH of about 5.5 to about 7. thing.
Item 16
Item 13. The pharmaceutical composition for use according to Item 13 or Item 14, wherein the pharmaceutical composition is formulated to deliver 3-HB or a salt thereof or a delivery means to the gastrointestinal tract 5 to 24 hours after oral administration after meals.
Item 17
Item 3. The pharmaceutical composition for the described use.
Item 18
The pharmaceutical composition for use according to any one of Items 3 to 17 or 3-HB for use according to Item 1 or Item 2, wherein the subject is a human.
Item 19
3-HB or item for use according to item 1 or item 2, wherein the inflammatory disease, disorder or condition is inflammatory bowel disease (IBD), preferably Crohn's disease or ulcerative colitis, or colorectal cancer. The pharmaceutical composition for use according to any one of 3-18.
Item 20
A method of treating an inflammatory disease, disorder or condition in a subject.
a) 3-Hydroxybutyric acid (3-HB) or a salt thereof;
b) Pharmaceutical composition comprising 3-HB or a salt thereof; and / or
c) Pharmaceutical composition comprising 3-HB delivery means
A method comprising administering.
Item 21
Substantially described herein as 3-hydroxybutyric acid (3-HB) or pharmaceutical composition.
Item 22
A composition comprising a genetically engineered anaerobic bacterium producing (R) -3-hydroxybutyrate and an orally ingestible carrier.
Item 23
Item 22. The composition according to Item 22, wherein the composition comprises an unnatural gene capable of expressing (R) -3-hydroxybutyryl-CoA dehydrogenase by a bacterium.
Item 24
Item 22. The composition according to Item 22 or Item 23, wherein the bacterium is a Clostridia bacterium.
Item 25
Item 6. The composition according to any one of Items 22 to 24, wherein the bacterium is from Clostridia taxa I, IV and / or XIVa.
Item 26
Item 6. The composition according to any one of Items 22 to 25, wherein the bacterium is derived from the genus Clostridium.
Item 27
Item 6. The composition according to any one of Items 22 to 26, wherein the bacterium is Clostridium butyricum.
Item 28
Item 6. The composition according to any one of Items 22 to 27, wherein the bacterium has a natural gene encoding phosphotransbutyrylase and / or butyrate kinase.
Item 29
Item 6. The composition according to any one of Items 22 to 28, wherein the bacterium produces (R) -3-hydroxybutyrate as the only fermentation product.
Item 30
Item 6. The composition according to any one of Items 22 to 29, wherein the bacterium produces (R) -3-hydroxybutyrate as a fermentation product in combination with acetate and / or butyrate.
Item 31
Item 6. The composition according to any one of Items 22 to 30, which comprises a pharmaceutically acceptable carrier for use as a pharmaceutical.
Item 32
Item 6. The composition according to any one of Items 22 to 31, for use in the treatment or prevention of gastrointestinal disorders.
Item 33
32. The composition according to Item 32, wherein the gastrointestinal disorder is inflammatory bowel disease or colon cancer.
Item 34
33. The composition according to Item 33, wherein the inflammatory bowel disease is selected from irritable bowel syndrome, Crohn's disease, cystitis, diverticulitis and ulcerative colitis.
Item 35
Item 6. The composition according to any one of Items 22 to 31, for use in the treatment of gastrointestinal bacterial flora abnormalities.
Item 36
Item 6. The composition according to any one of Items 22 to 31, for use in the treatment or prevention of Clostridium difficile infection.
Item 37
Item 6. The composition according to any one of Items 22 to 31, which is used for the regulation of the intestinal flora in a subject.
Item 38
Item 6. The composition according to any one of Items 22 to 31, which is used for feeding animals.
Item 39
Item 6. The composition according to any one of Items 22 to 31, which is in the form of capsules, tablets or powders.
Item 40
Item 6. The composition according to any one of Items 22 to 30, for use in foods.
Item 41
Item 40. The composition according to Item 40, wherein the food is a beverage, a drink, a dietary supplement or a functional food.
Item 42
Item 6. The composition according to any one of Items 22 to 41, wherein the Clostridium species is in the form of spores or vegetative cells.
Item 43
Item 6. The composition according to any one of Items 22 to 42, wherein the Clostridium species may be present in an amount of 1 × 10 ⁶ to 1 × 10 ¹⁰ CFU / composition g.
Item 44
(R) A method for treating or preventing a gastrointestinal disease or disorder comprising administering a genetically engineered anaerobic bacterium that produces -3-hydroxybutyrate.
Item 45
(R) A method for treating or preventing gastrointestinal flora abnormalities, comprising administering a genetically engineered anaerobic bacterium that produces -3-hydroxybutyrate.
Item 46
(R) A method for treating or preventing Clostridium difficile infection, comprising administering a genetically engineered anaerobic bacterium that produces -3-hydroxybutyrate.
Item 47
(R) A method of regulating the intestinal flora in a subject comprising administering a genetically engineered anaerobic bacterium that produces -3-hydroxybutyrate.
Item 48
47. The method of item 47, comprising administering to the animal a bait or food additive comprising the bacterium.
Item 49
Item 6. The method according to any one of Items 42 to 48, wherein the bacterium is Clostridia.
Item 50
Item 6. The method according to any one of Items 42 to 49, wherein the bacterium comprises an unnatural gene capable of expressing (R) -3-hydroxybutyryl-CoA dehydrogenase.
Item 51
Item 6. The method according to any one of Items 42 to 50, which comprises administering the bacterium orally.
Item 52
Item 6. The method according to any one of Items 42 to 51, further comprising prebiotic.

Claims (13)

Genetically engineered anaerobic bacteria and oral to produce (R) -3-HB for delivery of (R) -3-hydroxybutyrate ( (R) -3-HB) into the lumen of the gastrointestinal tract. A composition comprising an ingestible carrier , wherein the bacterium is found in the intestine, naturally produces butyrate, has a natural gene encoding phosphotransbutyrylase and butyrate kinase, and has (R) -3-hydroxy. A composition comprising an unnatural gene capable of expressing butyryl-CoA dehydrogenase . The composition according to claim 1 , wherein the bacterium is a Clostridia bacterium . The composition according to claim 1 or 2, wherein the bacterium is of a Clostridia taxon I, IV and / or XIVa . The composition according to any one of claims 1 to 3, wherein the bacterium is derived from the genus Clostridium. The composition according to any one of claims 1 to 4, wherein the bacterium is Clostridium butyricum . Claim 1 the bacterium produces (R) -3-hydroxybutyrate as the only fermentation product, or the bacterium produces (R) -3-hydroxybutyrate as a fermentation product in combination with acetate and / or butyrate. The composition according to any one of 5 to 5 . The composition according to any one of claims 1 to 6 , comprising a pharmaceutically acceptable carrier for use as a pharmaceutical. The composition according to any one of claims 1 to 7 , for use in a method of treating an inflammatory disease, disorder or condition in a subject. For use in the treatment or prevention of gastrointestinal disorders, if desired, the gastrointestinal disorders are inflammatory bowel disease or colon cancer, and the inflammatory bowel diseases are irritable bowel syndrome, Crohn's disease, cystitis, diverticulitis and ulcerative colitis. The composition according to any one of claims 1 to 8 , which is selected from flames. The composition for use according to any one of claims 7 to 9 , wherein the composition is for oral administration. 10. The composition of claim 10 , wherein the composition delivers (R) -3-HB to the large intestine, preferably the anaerobic portion of the large intestine, preferably the colon and / or the terminal ileum. a) Treatment of gastrointestinal bacterial flora abnormalities;
b) Treatment or prevention of Clostridium difficile infection c) Regulation of the intestinal flora in the subject or d) The composition according to any one of claims 1 to 6 for use in feeding animals. The composition according to any one of claims 1 to 7, for use in foods, wherein the food is a drink , a dietary supplement or a functional food.

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